CN117919511A - Preparation method of collagen matrix biological film, collagen matrix biological film and application thereof - Google Patents
Preparation method of collagen matrix biological film, collagen matrix biological film and application thereof Download PDFInfo
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Abstract
The application relates to a collagen matrix biological film preparation method, a collagen matrix biological film and application thereof, wherein the collagen matrix biological film preparation method comprises the following steps: s1: preparing a decellularized matrix layer with a compact structure; s2: preparing a collagen suspension; s3: extruding and injecting the collagen suspension into a mould for molding, then wetting the acellular matrix layer, spreading the wetted acellular matrix layer on the surface of the collagen suspension, performing light-pressure extrusion and bubble foaming, and standing to tightly combine the wetted acellular matrix layer with the collagen suspension; s4: freeze-drying the tightly combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer; s5: and (3) performing physical crosslinking on the composite freeze-dried layer to obtain the collagen matrix biomembrane. The collagen matrix biomembrane prepared by the preparation method has the advantages of high mechanical strength, good volume stability, good hydrophilicity, good adhesion with soft tissues of a receptor, capability of creating a microenvironment favorable for tissue healing, promotion of soft tissue regeneration and the like.
Description
Technical Field
The application relates to the technical field of biological materials, in particular to a preparation method of a collagen matrix biological film, the collagen matrix biological film and application thereof.
Background
Aiming at the problem of insufficient periodontal soft tissue, the most commonly adopted treatment method is periodontal soft tissue incremental operation. However, soft tissues required for soft tissue augmentation surgery often originate from autologous free gingival/connective tissue petals, have limited sources, have a relatively large postoperative patient response, and have a relatively narrow surgical scope, for which the heat of development of soft tissue replacement materials is high.
In the prior art, soft tissue replacement materials such as decellularized dermal matrix (acellular dermal matrix, ADM) represented by AlloDerm and mucoderm, and xenogeneic collagen matrix (xenogeneic collagen matrix, XCM) represented by Mucograft are most typical. Although evidence suggests that ADM is the closest graft substitute for connective tissue grafts (connective tissue graft, CTG), long-term follow-up results indicate that the therapeutic effect achieved with ADM suffers from poor gingival margin stability, possibly associated with its inability to induce epithelial cytokeratinization. XCM is a commercially available product in China, and has the product name of collagen matrix biofilm Mucograft, which has a double-layer structure, one layer has a compact structure, high strength and can be sutured, and allows wound protection in an open healing environment, and the second layer structure is a porous collagen sponge formed by freeze-drying collagen fiber slurry, so that blood clots can be stabilized. To avoid the introduction of additional crosslinking substances, ensure the biocompatibility of the product, mucograft employs an uncrosslinked design (EP 1252903B 1). From a biological point of view, like autologous connective tissue grafts, the purpose of XCM is to maintain space for fibroblast colonization from surrounding soft tissue, and then the matrix is gradually absorbed and replaced by autologous soft tissue. Gingival cells in connective tissue in the oral cavity are subjected to complex mechanical forces during chewing, speech, tooth movement and orthodontic treatment, and particularly during wound healing after surgery, internal and external forces may be generated, creating stress on newly formed tissues. Thus, the volume stability of the graft substitute is very important, also meaning that crosslinking of the material is necessary.
Through analysis of Mucograft products, the sponge layer is prepared by freeze drying of purified collagen fiber slurry, and has poor biological stability and mechanical properties due to no cross-linked structure, and particularly the bonding strength among fibers of the sponge layer is obviously reduced after water absorption, so that the collapse phenomenon can occur when pressure is applied. Thus adversely affecting the stability of the blood clot during the complex movements of the oral cavity. For the limitations of Mucograft products, researchers add crosslinking on the basis of the original products, and further develop second generation materials which have a thick single-layer sponge structure and have very good volume stability by adopting a chemical crosslinking method (application number 201580067861.4). Because of adopting a single-layer sponge structure and no strength support of a compact layer, the suture strength is low, and the clinical suture requirement cannot be met. In addition, the introduction of chemical cross-linking agents (such as glutaraldehyde, EDC-NHS) can reduce biocompatibility, adversely affect soft tissue healing, and affect tissue integration.
As noted in the text "A Bioreactor Test System to mimic the Biological and Mechanical Environment of Oral Soft Tissues and to Evaluate Substitutes for Connective Tissue Gafts" by Mathes et al, the soft tissue augmentation product requires as much balance of mechanical stability (high degree of cross-linking) as possible and smooth soft tissue healing (low degree of cross-linking) to achieve optimal soft tissue repair.
The best method for reinforcing the collagen material is to introduce a crosslinking structure, which is mainly divided into physical crosslinking and chemical crosslinking, wherein the physical crosslinking does not introduce additional chemical substances, but has the problems of uneven crosslinking and low crosslinking degree; although chemical crosslinking can improve the volume stability of the collagen material and prolong the degradation time, the residual crosslinking agent often causes inflammatory reaction of surrounding tissues, and as the material is degraded, the crosslinking agent is also released to continuously stimulate the surrounding tissues, so that the healing of the tissues is not facilitated. Moreover, chemical crosslinking often achieves too high a degree of crosslinking, which results in slow degradation of the material, and literature studies have indicated that excessive prolongation of degradation time can adversely affect tissue healing.
In this regard, studies on how to obtain an optimal soft tissue repair effect have been continued, for example, in the following related patents:
the Chinese patent application No. 202210620404.5 discloses an oral repair film and a preparation method thereof, wherein the oral repair film comprises a composite extracellular matrix layer and a collagen sponge layer arranged on the composite extracellular matrix layer, and the collagen sponge layer is prepared by freeze drying collagen gel prepared from crushed extracellular matrix powder or purified collagen powder. Wherein the extracellular matrix powder or the purified collagen powder is prepared by digestion and purification of protease, so that the natural cross-linked structure of the material is destroyed, and the strength of the material is influenced. In addition, the material is not crosslinked, can collapse after absorbing water, and has poor volume stability.
The Chinese patent application No. 202310655636.9 discloses a pig peritoneum decellularized matrix sponge scaffold and a preparation method thereof, wherein the pig peritoneum is degreased and decellularized, and the sponge scaffold is prepared through the processes of crushing, enzymolysis, purification, crosslinking and the like, and the decellularized matrix is digested by protease, so that the natural crosslinking structure of collagen fibers is damaged, the overall strength of the material is reduced, and the degradation rate is too high; and the strength of the material is enhanced by chemical crosslinking, so that the biocompatibility of the material is reduced.
Chinese patent application No. 202211143378.8 discloses a collagen matrix flap with stable wet volume and a preparation method thereof, sodium periodate is added into sodium alginate solution, oxidized sodium alginate is prepared by reaction, then oxidized sodium alginate solution is added into the collagen solution drop by drop, chemical crosslinking is introduced, and then the collagen matrix flap with excellent wet volume stability is obtained by dialysis and freeze-drying. The introduction of multi-step chemical reactions and additional components in the material preparation process increases the risks in practical applications.
In summary, the prior art cannot obtain a repair material with specific volume stability and stable soft tissue healing performance, and most of the repair materials have the defects of poor volume stability, too fast degradation rate or low biocompatibility. On the other hand, the current domestic collagen sponge product cannot meet the requirement of long-time volume stability, and most of clinically applied soft tissue substitute materials are derived from abroad, so that the cost is very high, and the economic burden of patients is increased.
Therefore, there is an urgent need to develop a soft tissue augmentation bio-substitute material with better volume stability and biocompatibility, which not only shortens the operation time and reduces the postoperative complications, but also is expected to achieve clinical therapeutic effects comparable to or even better than the application of autologous soft tissue grafts.
Disclosure of Invention
In view of the above, the present application aims to provide a preparation method of a collagen matrix biofilm, a collagen matrix biofilm and an application thereof, so as to solve the disadvantages of poor volume stability, poor mechanical strength, material variability and difficulty in fully matching with a patient injury of a soft tissue increment biological replacement material in the prior art.
The invention provides a preparation method of a collagen matrix biological membrane, which comprises the following steps:
s1: preparing a decellularized matrix layer with a compact structure;
S2: preparing a collagen suspension;
S3: extruding and injecting the collagen suspension into a mould for molding, then wetting the acellular matrix layer, spreading the wetted acellular matrix layer on the surface of the collagen suspension, performing light-pressure extrusion and bubble foaming, and standing to tightly combine the acellular matrix layer with the collagen suspension;
s4: freeze-drying the tightly combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer;
S5: and (3) performing physical crosslinking on the composite freeze-dried layer to obtain the collagen matrix biomembrane.
Further, the acellular matrix layer with the compact structure is a multi-layer composite acellular matrix layer, wherein S1 further comprises the following steps,
S11, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S12 compounding: and carrying out vacuum pressing compounding on the acellular matrix of 2-6 layers to obtain a multi-layer composite acellular matrix layer.
Further, the decellularized matrix layer having a dense structure is a recombinant decellularized matrix layer, wherein S1 further comprises the steps of,
S101, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S102, crushing: crushing the acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5-3.0%, and adjusting the pH value to 2.5-3.0 by using acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension;
S103, dehydration: adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0-2.5mol/L to obtain white salting-out precipitate; soaking and dehydrating the white salting-out precipitate with an organic solvent for 0.5-2h, rinsing with PBS buffer solution, and removing the organic solvent to obtain collagen fiber;
S104, dispersing: suspending collagen fibers in PBS buffer solution to make the mass percentage of the collagen fibers be 0.1% -6%, and mechanically stirring to obtain uniformly dispersed and fluffy collagen fiber suspension;
S105, film forming: and (3) passing the collagen fiber suspension through a vacuum suction filtration device to ensure that the collagen fibers are uniformly trapped on a filter membrane with the aperture of 0.8 mu m, continuing suction filtration until the collagen fibers are in a dry semitransparent film shape, and stripping to obtain the recombinant acellular matrix layer with the thickness of 0.1-0.5 mm.
Further, the collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, 5-30 parts of gel-like collagen liquid and 70-95 parts of collagen fiber liquid are mixed, stirred uniformly and vacuumed to obtain the collagen suspension.
Further, the preparation step of the gel-like collagen liquid comprises the following steps:
s201 decellularization: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S202, crushing: cutting acellular matrix, suspending in pure water to make the mass percentage of acellular matrix be 0.5% -3.0%, adjusting pH to 3.0-4.0 with acid, and pulverizing acellular matrix to gel with tissue masher to obtain gel collagen liquid.
Further, the preparation step of the collagen fiber solution comprises the following steps:
S211, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S212, crushing: crushing the acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5-3.0%, and adjusting the pH value to 2.5-3.0 by using acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension;
S213, dehydration: adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0-2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, and centrifuging to obtain concentrated collagen fiber;
S214 dispersion: placing the collagen fibers in acid with pH value of 3.0-4.0 to make the mass percentage of the collagen fibers be 1% -6%, and mechanically stirring to make the collagen fibers present slightly swollen uniform dispersion so as to obtain collagen fiber liquid.
Further, the naturally derived tissue comprises porcine or bovine small intestine submucosa, porcine or bovine peritoneum, porcine or bovine pericardium, porcine or bovine bladder, porcine or bovine dermis.
Further, the organic solvent comprises ethanol, acetone, diethyl ether and isopropanol.
Further, the wet acellular matrix layer in step S3 is obtained by immersing the acellular matrix layer in an acid having a pH of 3.0 to 4.0 for 10 to 30 minutes.
Further, the acid comprises hydrochloric acid, acetic acid, citric acid.
Further, the physical crosslinking in the step S5 is gradient thermal crosslinking, the composite freeze-dried layer is placed into a vacuum box, vacuumized to be less than-100 Kpa, and kept at 25-30 ℃ for 1-2 h; crosslinking by gradient heating, heating to 80-85 ℃, and preserving heat for 2-3h; continuously heating to 100-105 ℃, and preserving heat for 2-3h; heating to 120-125 deg.c and maintaining for 24-48 hr.
The invention also provides a collagen matrix biomembrane, which is prepared by the preparation method.
The invention also provides an application scene of the collagen matrix biological film, which comprises the steps of applying the collagen matrix biological film prepared according to the preparation method to any one or more of the following scenes: the stomatology guides bone tissue regeneration, the increment of the stomatology soft tissue, prevents tissue adhesion and chronic wound repair.
The invention has the beneficial effects that:
According to the collagen matrix biological membrane provided by the invention, the acellular matrix is prepared by taking natural source animal tissues as raw materials, and then the acellular matrix is further processed to obtain the collagen matrix biological membrane with a compact layer and loose sponge layer double-layer structure.
(1) The compact layer carries out matching design on the structure according to different animal tissue sources; when small intestine of pig or cow or bladder tissue of pig or cow is selected, the compact layer adopts the design of multi-layer composite decellularized matrix layer, so that the mechanical strength and degradability of the compact layer are improved. When the pig or cattle peritoneum, the pig or cattle pericardium and the pig or cattle dermis are selected, the compact layer adopts the design of a recombinant acellular matrix layer, so that the degradability is improved and a certain mechanical strength is maintained.
(2) The loose layer is lyophilized from a collagen suspension obtained from a gelatinous collagen solution and a collagen fiber solution, and further processed from a acellular matrix. The gel-like collagen liquid is a viscoelastic gel state system prepared by sufficiently crushing acellular matrix, the system is uniform and has more physical entanglement, and a structure with a relatively uniform pore structure can be obtained after freeze-drying, but the gel-like collagen liquid has low mechanical strength and is easy to degrade due to the fact that most of natural connections among fibers are broken; the acellular matrix particles used in the preparation of the collagen fiber liquid are larger, more natural structures are maintained, the mechanical strength is high, and the degradation time is longer. However, when the collagen fiber solution is prepared into a freeze-dried sponge alone, the sponge structure is not uniform and is easy to collapse after absorbing water, because of the low action strength among the collagen fibers. Therefore, the gel-like collagen liquid and the collagen fiber liquid are mixed according to a certain proportion, wherein the gel-like collagen liquid also plays a role of a thickening agent or a binding agent, and the interaction between the collagen fibers is enhanced, so that the finally obtained sponge structure has the mechanical property and the property of a uniform pore structure, and the phenomenon of collapsibility is not easy to occur after liquid absorption. Meanwhile, the gel-like collagen liquid is added to enable the connection between the compact layer and the loose sponge layer to be more compact.
(3) In the preparation process, the wet acellular matrix layer is flatly paved on the surface of the collagen suspension, the air bubbles are compressed under light pressure, and the acellular matrix layer is tightly combined with the collagen suspension by standing. The gel-like collagen liquid is added to enhance the viscosity of the collagen suspension, so that more physical entanglement acting force is generated between the acellular matrix layer and the collagen suspension, and the compact layer and the loose sponge layer are tightly combined together mainly through physical entanglement and hydrogen bonding.
(4) The collagen matrix biological membrane is subjected to physical crosslinking treatment, so that the interaction among collagen fibers is increased, the volume stability of the collagen matrix biological membrane is better, and good biocompatibility is ensured. The collagen matrix membrane which is reasonably designed and processed in the invention overcomes the defects of poor biostability and mechanical property in the prior art, and the collagen matrix membrane which is subjected to physical entanglement and physical crosslinking treatment has stable mechanical property after absorbing water, and has no macroscopic collapse phenomenon after applying pressure. The collagen matrix membrane is a soft tissue increment biological substitute material with better volume stability and biocompatibility, can be better matched with the wound of a patient, and promotes healing.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic cross-sectional view of a collagen matrix biofilm according to the present application;
FIG. 2 is a scanning electron microscope image of a cross-sectional structure of a collagen matrix biofilm prepared in example 1;
FIG. 3 is a graph comparing the results of the wet volume stability test of the collagen matrix biofilm and Mucograft products of example 1.
Reference numerals in the figures
1-Compact layer, 2-sponge layer.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a preparation method of a collagen matrix biological membrane, which comprises the following steps:
s1: preparing a decellularized matrix layer with a compact structure;
S2: preparing a collagen suspension;
S3: extruding and injecting the collagen suspension into a mould for molding, then wetting the acellular matrix layer, spreading the wetted acellular matrix layer on the surface of the collagen suspension, performing light-pressure extrusion and bubble foaming, and standing to tightly combine the acellular matrix layer with the collagen suspension;
s4: freeze-drying the tightly combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer;
S5: and (3) performing physical crosslinking on the composite freeze-dried layer to obtain the collagen matrix biomembrane.
The collagen matrix biomembrane obtained by the preparation method has a double-layer structure, and one layer of the collagen matrix biomembrane is a cell-free matrix layer with a compact structure, so that the mechanical strength capable of being sutured can be provided, and meanwhile, the wound can be protected in an open healing environment; the other layer is a loose sponge layer which is prepared by freeze drying of collagen suspension, and the loose layer has a uniform pore structure, can quickly absorb blood and body fluid, stabilize blood clots and promote tissue healing. The collagen matrix membrane is a soft tissue increment biological substitute material with better volume stability and biocompatibility through structural design and matching on macroscopic and microscopic scales, can be matched with the wounded parts of patients better, and promotes healing.
In the invention, the acellular matrix layer with a compact structure is a multi-layer composite acellular matrix layer or a recombinant acellular matrix layer, and the acellular matrix is animal tissue of natural origin and is prepared by an acellular process.
In some preferred embodiments, the naturally derived animal tissue may be one of porcine or bovine small intestine submucosa, porcine or bovine peritoneum, porcine or bovine pericardium, porcine or bovine bladder, porcine or bovine dermis.
In some preferred embodiments, when the densely structured decellularized matrix layer is selected from the group consisting of natural source animal tissue that is porcine or bovine small intestine submucosa, porcine or bovine bladder, it is desirable to prepare a multi-layered composite decellularized matrix layer, i.e., the densely structured decellularized matrix layer is a multi-layered composite decellularized matrix layer, wherein S1 further comprises the steps of:
S11, cell removal: pretreating natural animal tissue, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S12 compounding: and carrying out vacuum pressing compounding on the multi-layer acellular matrix to obtain a multi-layer composite acellular matrix layer.
In some preferred embodiments, the number of layers of decellularized matrix is preferably 2-6 for the purpose of regulating mechanical strength and degradation time, as the porcine or bovine small intestine submucosa, porcine or bovine bladder becomes very thin after decellularization treatment.
It should be noted that one of the technical problems to be solved in the present invention is "a repair material combining specific volume stability and smooth soft tissue healing properties", that is, "a balance between achieving mechanical stability (high degree of crosslinking) and smooth soft tissue healing (low degree of crosslinking)" mentioned in the background art. The single-layer pig or cow small intestine submucosa and pig or cow bladder acellular matrix are very thin, the thickness is 0.02-0.03mm, and in order to obtain better degradability and mechanical strength, 6-12 layers of acellular matrix composites are adopted in products at home and abroad, and meanwhile, the natural collagen fiber structure without cross-linking is maintained. Since the collagen matrix biofilm of the present invention is subjected to physical crosslinking treatment, in order to avoid the degradation of the acellular matrix of the urinary tract submucosa of a pig or a cow, or the urinary bladder of the pig or the cow, which is difficult to degrade, and the tissue healing is affected, the acellular matrix composite layer of the present invention is preferably 2-6 layers, more preferably 3-5 layers.
In some preferred embodiments, when the natural source animal tissue selected for the dense structure acellular matrix layer is one of porcine or bovine peritoneum, porcine or bovine pericardium, porcine or bovine dermis, it is necessary to recombine the acellular matrix layer to prepare a recombined acellular matrix layer, i.e. when the acellular matrix layer with dense structure is a recombined acellular matrix layer, wherein S1 further comprises the steps of: :
S101, cell removal: pretreating pig or cattle peritoneum, pig or cattle pericardium, pig or cattle dermis tissue, inactivating virus, degreasing, decellularizing and drying to obtain decellularized matrix;
s102, crushing: crushing the acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5-3.0%, and adjusting the pH value of the pure water to 2.5-3.0 by using acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension;
in some preferred embodiments, the acid is preferably one of hydrochloric acid, acetic acid, citric acid;
S103, dehydration: adding sodium chloride powder into the fully swelled decellularized matrix suspension until the concentration of sodium chloride reaches 2.0-2.5mol/L to obtain white salting-out precipitate, soaking and dehydrating the white salting-out precipitate with an organic solvent for 0.5-2h, and rinsing with PBS buffer solution (phosphate buffer solution) to obtain collagen fiber;
in some preferred embodiments, the organic solvent may be one of ethanol, acetone, diethyl ether, and isopropanol.
S104, dispersing: suspending the collagen fibers in PBS buffer solution to make the mass percentage of the collagen fibers be 0.1-6%, and mechanically stirring to disperse the uniformly fluffy collagen fiber suspension;
s105, film forming: and (3) passing the collagen fiber suspension through a vacuum suction filtration device to uniformly intercept the collagen fibers on a filter membrane with the aperture of 0.8 mu m, continuing suction filtration until the collagen fibers are in a dry semitransparent film shape, stripping to obtain a collagen fiber membrane with the thickness of 0.1-0.5mm, and rinsing with pure water to obtain a recombinant acellular matrix layer.
It should be noted that, in the preparation of the recombinant acellular matrix layer, one of the technical problems to be solved in the present invention is "a repair material which combines specific volume stability and smooth soft tissue healing properties". The acellular matrix of the peritoneum of the pig or the cattle, the pericardium of the pig or the cattle and the dermis of the pig or the cattle is thicker, and the single-layer structure without cross-linking can generally meet the clinical use requirement. Considering that the collagen matrix biological membrane in the invention needs to be subjected to physical crosslinking treatment, in order to avoid the introduction of crosslinking, the porcine or bovine peritoneum, the porcine or bovine pericardium and the porcine or bovine dermis acellular matrix are difficult to degrade and influence the tissue healing, and the recombinant acellular matrix layer is prepared in the invention. In the preparation step of the recombinant acellular matrix layer, acellular matrix particles with the preferred particle size range can be fully swelled in acid with the preferred pH range, and loose white collagen fibers can be obtained through salting out and precipitation treatment; then, the collagen fiber is dehydrated and contracted by the treatment of an organic solvent, so that the mechanical strength and the swelling resistance of the collagen fiber are enhanced, and the phenomenon that the final recombined decellularized matrix layer is excessively swelled when contacting water is avoided; washing the dehydrated collagen fibers with PBS buffer solution for a plurality of times to remove the organic solvent; the collagen fibers are resuspended in PBS buffer solution to make the mass percentage of the collagen fibers be 0.1% -6%, and the collagen fibers are not swelled in PBS, so that the evenly dispersed and fluffy collagen fiber suspension can be obtained by mechanical stirring. The series of treatment processes are organic whole, and the aim is to make the film forming process smoothly carried out; in order to avoid the blocking of the filter membrane pores by the collagen fibers, the collagen fibers are fully contracted by the treatment of an organic solvent, the swelling of the collagen fibers is avoided, and a large-aperture filter membrane with the diameter of 0.8 mu m is selected. Through negative pressure suction filtration, water passes through the filter membrane, and collagen fibers are trapped on the filter membrane, and the suction filtration is continued until the collagen fibers are in a dry semitransparent film shape, so that a collagen fiber membrane with a certain thickness is finally formed. In order to maintain reasonable mechanical strength and degradation time, the thickness of the collagen fiber membrane is preferably 0.06mm to 0.18mm.
The collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, wherein the gel-like collagen liquid and the collagen fiber liquid are prepared from acellular matrixes of natural animal tissues, and the natural animal tissues can be one of pig or cow small intestine submucosa, pig or cow peritoneum, pig or cow pericardium, pig or cow bladder, pig or cow dermis.
Specifically, the preparation method of the gel-like collagen liquid comprises the following steps:
s201 decellularization: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S202, crushing: cutting the acellular matrix subjected to the acellular treatment, suspending in pure water to make the mass percentage of the acellular matrix be 0.5% -3.0%, adjusting the pH value of the pure water to 3.0-4.0 by using acid, and grinding the acellular matrix to gel by using a tissue masher to obtain gel collagen liquid.
In some preferred embodiments, since the decellularized matrix is capable of swelling in an acidic solution, accompanied by the strong shearing and mixing effects of the tissue masher, the decellularized matrix is crushed into very small particles and further swelled, such that the solution viscosity gradually increases, ultimately obtaining a viscous gel-like collagen solution.
In some preferred embodiments, the acid may be one of hydrochloric acid, acetic acid, and citric acid.
In some preferred embodiments, the mass percentage of acellular matrix in pure water is preferably 0.5% -3.0% because when the mass percentage of acellular matrix is less than 0.5%, the final collagen solution assumes a flowing solution state, and the viscosity is low, which can adversely affect the subsequent preparation process of the collagen suspension; when the mass percentage of the acellular matrix is more than 3.0%, the final collagen liquid loses fluidity due to the excessively high concentration, so that part of the swelling particles cannot be sufficiently crushed, and the crushing effect is affected; the viscosity of the gel-like collagen liquid to be finally obtained is dependent on the pH value, and the smaller the pH value, the larger the viscosity, and the pH value is preferably in the range of 3.0 to 4.0 in order to avoid the influence of the excessive viscosity of the gel-like collagen liquid on the pulverizing effect.
In some preferred embodiments, the collagen fiber solution is prepared as follows:
S211, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S212, crushing: crushing the acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5-3.0%, and adjusting the pH value of the pure water to 2.5-3.0 by using acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension;
s213, dehydration: adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0-2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber;
S214 dispersion: placing the collagen fibers in acid with the pH value of 3.0-4.0 to enable the mass percentage of the collagen fibers to be 1-6%, and enabling the collagen fibers to be in a slightly-swelled uniform dispersion state through mechanical stirring to obtain collagen fiber liquid; the acid can be one of hydrochloric acid, acetic acid and citric acid.
In the preparation process of the collagen fiber liquid, crushed acellular matrix particles are swelled in an acid solution with the pH value of 2.5-3.0, and loose white collagen fibers can be obtained through salting out and precipitation treatment; in order to maintain the natural structure of the acellular matrix to some extent, therefore, the particle size of the acellular matrix after pulverization is preferably 0.2mm to 2mm.
In some preferred embodiments, to avoid delamination of the collagen fibril solution, the collagen fibrils are dispersed in an acid solution having a pH of 3.0-4.0, allowing the collagen fibrils to slightly swell, and allowing the collagen fibrils to uniformly disperse by mechanical agitation.
In some preferred embodiments, the pH of the collagen fibrillar fluid should be the same as the pH of the gelatinous collagen fluid in order to match the gelatinous collagen fluid.
The preparation process of the collagen fiber solution is different from the preparation process of the recombinant acellular matrix layer, in that the salted-out collagen fiber is not dehydrated by an organic solvent. The organic solvent treatment can dehydrate the collagen fibers to increase the mechanical properties, but can reduce the swelling effect in the acid solution. In addition, the purpose of preparing the collagen fiber solution is to compound with the gel-like collagen solution to prepare a uniform and loose porous sponge layer, and the recombinant acellular matrix layer is more prone to mechanical properties, so that the organic solvent dehydration treatment is not needed here.
After the gel-like collagen liquid and the collagen fiber liquid are respectively prepared, the gel-like collagen liquid and the collagen fiber liquid are mixed according to a proportion, stirred uniformly and vacuumized to remove bubbles, so as to obtain a collagen suspension.
In some preferred embodiments, 5-30 parts of gel-like collagen liquid and 70-95 parts of collagen fiber liquid are taken, and the gel-like collagen liquid and the collagen fiber liquid are uniformly stirred and vacuolated to obtain collagen suspension.
In some preferred embodiments, since the gel-like collagen solution is a viscoelastic gel-state system prepared by sufficiently pulverizing the acellular matrix, the system is uniform and has more physical entanglement, and a structure with a relatively uniform pore structure can be obtained after freeze-drying, but the gel-like collagen solution has low mechanical strength and is easy to degrade due to the destruction of most of natural links among fibers; the acellular matrix particles used in the preparation of the collagen fiber liquid are larger, more natural structures are maintained, the mechanical strength is high, and the degradation time is longer. However, when the collagen fiber solution is prepared into a freeze-dried sponge alone, the sponge structure is uneven, and the collagen fiber solution is easy to collapse after being subjected to pressure, because of the low action strength among the collagen fibers. Therefore, the gel-like collagen liquid and the collagen fiber liquid are mixed according to a certain proportion, wherein the gel-like collagen liquid also plays a role of a thickening agent or a binding agent, so that the finally obtained sponge structure has the mechanical property and the property of a uniform pore structure, and the phenomenon of collapsibility is not easy to occur after liquid absorption. Meanwhile, the gel-like collagen liquid is added to enable the connection between the compact layer and the loose sponge layer to be more compact.
Further, extruding and injecting the collagen suspension into a mould for forming, immersing the acellular matrix layer in an acid solution with the pH value of 3.0-4.0 for 10-30min to obtain a wet acellular matrix layer, spreading the wet acellular matrix layer on the surface of the collagen suspension, performing light pressure foaming, and standing to tightly combine the wet acellular matrix layer with the collagen suspension. The gel-like collagen liquid is added to enhance the viscosity of the collagen suspension, so that more physical entanglement force is generated between the acellular matrix layer and the collagen suspension, and the compact layer and the loose sponge layer are tightly combined together mainly through physical entanglement and hydrogen bonding.
In some preferred embodiments, the collagen matrix biofilm of the present application is obtained by physical crosslinking. The physical crosslinking is gradient thermal crosslinking, the composite freeze-dried layer is placed into a vacuum box, vacuumized to be less than 100Kpa, and kept at 25 ℃ to 30 ℃ for 1h to 2h; crosslinking by gradient heating, heating to 80-85 ℃, and preserving heat for 2-3h; continuously heating to 100-105 ℃, and preserving heat for 2-3h; heating to 120-125 deg.c and maintaining for 24-48 hr. The collagen matrix biological membrane is subjected to physical crosslinking treatment, so that the interaction among collagen fibers is increased, the volume stability of the collagen matrix biological membrane is better, and good biocompatibility is ensured.
As described in the background art, the sponge layer of Mucograft products on the market is a collagen fiber structure after lyophilization, which collapses after absorbing water and pressurizing. In the invention, more physical entanglement is introduced and gradient thermal crosslinking treatment is carried out, so that the interaction strength among collagen fibers is greatly enhanced, and the volume stability and degradability of the sponge layer are fundamentally improved. In the invention, the collagen fibers in the collagen fiber liquid are contracted due to the swelling and salting-out processes, and the freeze-dried sponge is easy to collapse and collapse after imbibition due to the lack of connection among the fibers. To solve this problem, a gradient thermal crosslinking treatment is introduced in the present technology. The gradient thermal crosslinking is a physical crosslinking, and in the crosslinking process, the carboxyl and the amino of the side chains of adjacent amino acids can be subjected to condensation reaction to form ester bonds and amide bonds, so that the three-helix structure of collagen is not excessively damaged by the crosslinking mode, and an additional chemical crosslinking agent is not introduced. However, in experiments, the contracted collagen fiber structure also means fewer active groups, and the crosslinking density is low after gradient thermal crosslinking, so that the purpose of improving the liquid absorption collapsibility cannot be achieved. Therefore, the technology further introduces gel-like collagen liquid, more physical entanglement structures exist in the gel-like collagen liquid, and after the gel-like collagen liquid is fully mixed with collagen fibers, the gel-like collagen liquid can act as a connecting bridge to increase the interaction among the collagen fibers, so that the collagen fibers are connected into a whole. After gradient thermal crosslinking, more ester bonds and amide bonds are formed, so that the obtained collagen matrix biomembrane does not have obvious collapsibility phenomenon after imbibition, and has excellent volume stability.
The invention also provides the collagen matrix biological membrane prepared by the method, which comprises a compact layer 1 and a loose sponge layer 2, wherein the compact layer 1 comprises 2-6 layers of composite acellular matrixes or recombinant acellular matrix layers, the compact layer 1 and the loose sponge layer 2 are tightly connected through physical entanglement, hydrogen bonding and physical crosslinking, and the loose sponge layer 2 is used for being tightly adhered to a soft tissue defect area;
the loose sponge layer 2 is a loose collagen layer with pores, and the loose sponge layer 2 comprises a gel-like collagen liquid freeze-dried layer and a collagen fiber liquid freeze-dried layer.
In some preferred embodiments, the loose sponge layer 2 is obtained by mixing gel-like collagen liquid and collagen fiber liquid and freeze-drying the mixture, and the gel-like collagen liquid freeze-dried layer and the collagen fiber liquid freeze-dried layer are mutually interwoven. Physical crosslinking is adopted to further strengthen the mechanical strength and the volume stability of the collagen matrix biomembrane. In use, the dense layer 1 provides a reliable suturing force and allows for open wound healing; the loose sponge layer 2 faces to the soft tissue defect area, so that blood clots can be stabilized, and tissue repair is promoted. The loose sponge layer 2 has excellent hydrophilicity, so that the loose sponge layer can quickly absorb body fluid at the tissue defect part and further closely adhere to the tissue defect part.
The application also provides an application of the collagen matrix biological membrane, which comprises the steps of applying the collagen matrix biological membrane prepared by the preparation method to any one or more of the following scenes: the stomatology guides bone tissue regeneration, the increment of the stomatology soft tissue, prevents tissue adhesion and chronic wound repair.
In order to further illustrate the present invention, a collagen matrix biofilm and a method for preparing the same according to the present invention will be described in detail with reference to examples.
The raw materials and reagents used in the following examples are all commercially available.
Example 1
The naturally derived tissue in this example was selected from porcine small intestine submucosa, and acellular matrix was first prepared. Removing redundant tissues from the small intestine of a pig, soaking the small intestine of the pig in 0.1% peracetic acid solution for 1 hour, and washing the small intestine of the pig with running water; shake-cleaning with 0.05% trypsin and 0.05% sodium dodecyl sulfate mixed solution for 1 hr, and cleaning with running water; soaking and degreasing the pig small intestine submucosa acellular matrix by using isopropanol with the mass percent of 75%, and freeze-drying to obtain the pig small intestine submucosa acellular matrix, wherein the steps of pretreatment, virus inactivation, degreasing, acellular, drying and the like are conventional steps in the prior art, and the specific contents are not further described herein.
The preparation of collagen matrix biofilm was performed as follows:
① Preparation of a dense structure acellular matrix layer: and carrying out vacuum pressing compounding on the 6 layers of pig small intestine submucosa acellular matrixes to obtain a plurality of layers of composite acellular matrixes.
② Preparation of collagen suspension:
Firstly, preparing gel-like collagen liquid, shearing acellular matrix of submucosa of small intestine of a pig, suspending in pure water to enable the mass percentage of the acellular matrix to be 1.0%, adjusting the pH value of the pure water to 3.0 by using acetic acid, and crushing the acellular matrix to gel-like by using a tissue masher to obtain the gel-like collagen liquid;
Then preparing collagen fiber liquid, grinding the pig small intestine submucosa acellular matrix to irregular particles with the particle size of 0.2mm-2mm through low-temperature grinding, suspending the particles in pure water to enable the mass percentage of the acellular matrix particles to be 3.0%, and adjusting the pH value to 3.0 through acetic acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber; placing the collagen fibers in acetic acid with the pH value of 3.0 to enable the mass percentage of the collagen fibers to be 3%, and mechanically stirring to enable the collagen fibers to be in a slightly-swollen and uniformly-dispersed state to obtain collagen fiber liquid;
the collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, and is prepared by mixing 20 parts of gel-like collagen liquid and 80 parts of collagen fiber liquid, uniformly stirring, and removing bubbles in vacuum.
③ Extruding and injecting the collagen suspension into a mould for forming, placing a plurality of layers of composite pig small intestine submucosa acellular matrix layers into acetic acid with the pH of 3.0 for wetting for 10min, spreading the wetted acellular matrix on the surface of the collagen suspension, performing light pressure extrusion and bubble foaming, and standing to tightly combine the acellular matrix layers with the collagen suspension;
Freeze-drying the closely combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer, putting the composite freeze-dried layer into a vacuum box, vacuumizing to-110 Kpa, and keeping at 25 ℃ for 2 hours; crosslinking by gradient heating, heating to 80 ℃, and preserving heat for 3h; continuously heating to 100 ℃, and preserving heat for 3 hours; heating to 120 ℃, and preserving heat for 48 hours. And cooling to normal temperature, and taking out the collagen matrix biomembrane.
Example 2
The naturally derived tissue in this example was selected from porcine pericardium, and first the acellular matrix was prepared. Removing redundant tissues from pig pericardium tissues, soaking the pig pericardium tissues in 0.1% peracetic acid solution for 1 hour, and washing the pig pericardium tissues with running water; shake-cleaning with 0.05% trypsin and 0.05% sodium dodecyl sulfate mixed solution for 1 hr, and cleaning with running water; soaking and degreasing the pig pericardium acellular matrix by using 75% of isopropanol by mass percent, and freeze-drying the pig pericardium acellular matrix.
The preparation of collagen matrix biofilm was performed as follows:
① Preparation of a dense structure acellular matrix layer: grinding pig pericardium acellular matrix to irregular particles with the particle size of 0.2-2 mm by low-temperature grinding, suspending in pure water to enable the mass percentage of acellular matrix particles to be 0.5%, and adjusting the pH value of the pure water to 2.5 by hydrochloric acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0mol/L to obtain white salting-out precipitate; soaking and dehydrating the white salting-out precipitate with acetone for 0.5h, rinsing with PBS buffer solution, and removing residual acetone to obtain collagen fiber; suspending collagen fibers in PBS buffer solution to make the mass percentage of the collagen fibers be 0.1%, and dispersing the collagen fibers into a uniformly fluffy collagen fiber suspension by mechanical stirring; and (3) passing the collagen fiber suspension through a vacuum suction filtration device to ensure that the collagen fibers are uniformly trapped on a filter membrane with the aperture of 0.8 mu m, continuing suction filtration until the collagen fibers are in a dry semitransparent film shape, and stripping to obtain a recombinant acellular matrix layer with the thickness of 0.1 mm.
② Preparation of collagen suspension: firstly, preparing gel-like collagen liquid, shearing pig pericardium acellular matrix, suspending in pure water to enable the mass percentage of the acellular matrix to be 0.5%, adjusting pH to 4.0 by acetic acid, and crushing the acellular matrix to gel-like by a tissue masher to obtain the gel-like collagen liquid;
Then preparing collagen fiber liquid, grinding pig pericardium acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending in pure water to enable the mass percentage of acellular matrix particles to be 3.0%, adjusting the pH value to 3.0 through acetic acid, and fully swelling the acellular matrix particles to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber; placing collagen fibers in acetic acid with a pH value of 4.0 to enable the mass percentage of the collagen fibers to be 6%, and mechanically stirring to enable the collagen fibers to be in a slightly-swollen and uniformly-dispersed state to obtain collagen fiber liquid;
The collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, and is prepared by mixing 5 parts of gel-like collagen liquid and 95 parts of collagen fiber liquid, uniformly stirring, and removing bubbles in vacuum.
③ Extruding and injecting the collagen suspension into a mould for forming, placing the recombinant pig pericardium acellular matrix layer into acetic acid with pH of 4.0 for wetting for 10min, spreading the wetted acellular matrix on the surface of the collagen suspension, performing light pressure extrusion and bubble generation, and standing to tightly combine the acellular matrix layer with the collagen suspension;
④ Freeze-drying the closely combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer, putting the composite freeze-dried layer into a vacuum box, vacuumizing to-110 Kpa, and keeping at 30 ℃ for 1h; crosslinking by gradient heating, heating to 85 ℃, and preserving heat for 2h; continuously heating to 105 ℃, and preserving heat for 2 hours; heating to 125 ℃, and preserving heat for 24 hours. And cooling to normal temperature, and taking out the collagen matrix biomembrane.
Example 3
In this example, porcine small intestine submucosa acellular matrix and porcine pericardium acellular matrix were prepared by the methods of examples 1 and 2.
The preparation of collagen matrix biofilm was performed as follows:
① Preparation of a dense structure acellular matrix layer: and carrying out vacuum pressing and compounding on the wet acellular matrix of the 2 layers of pig small intestine submucosa to obtain a multi-layer composite acellular matrix layer.
② Preparation of collagen suspension: firstly, preparing gel-like collagen liquid, shearing and suspending pig pericardium acellular matrix in pure water to enable the mass percentage of the acellular matrix to be 2%, adjusting the pH value to 4.0 by acetic acid, and crushing the acellular matrix to gel-like by a tissue masher to obtain the gel-like collagen liquid;
Then preparing collagen fiber liquid, grinding pig pericardium acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of acellular matrix particles to be 2.0%, and adjusting the pH value of the pure water to 2.5 through acetic acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber; placing collagen fibers in acetic acid with a pH value of 4.0 to enable the mass percentage of the collagen fibers to be 4%, and mechanically stirring to enable the collagen fibers to be in a slightly-swollen and uniformly-dispersed state to obtain collagen fiber liquid;
The collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, 10 parts of gel-like collagen liquid and 90 parts of collagen fiber liquid are mixed, stirred uniformly and vacuumed to obtain the collagen suspension.
③ Extruding and injecting the collagen suspension into a mould for forming, placing a plurality of layers of composite pig small intestine submucosa acellular matrix layers into acetic acid with pH of 4.0 for wetting for 30min, spreading the wetted acellular matrix on the surface of the collagen suspension, performing light pressure extrusion and bubble foaming, and standing to tightly combine the acellular matrix layers with the collagen suspension;
④ Freeze-drying the tightly combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer, putting the composite freeze-dried layer into a vacuum box, vacuumizing to less than-100 Kpa, and keeping at 30 ℃ for 1h; crosslinking by gradient heating, heating to 80 ℃, and preserving heat for 2h; continuously heating to 100 ℃, and preserving heat for 2 hours; heating to 120 ℃, and preserving heat for 36h. And cooling to normal temperature, and taking out the collagen matrix biomembrane.
Example 4
In this example, porcine small intestine submucosa acellular matrix and porcine pericardium acellular matrix were prepared by the methods of examples 1 and 2.
The preparation of collagen matrix biofilm was performed as follows:
① Preparation of a dense structure acellular matrix layer: grinding pig pericardium acellular matrix to irregular particles with the particle size of 0.2-2 mm by low-temperature grinding, suspending in pure water to enable the mass percentage of acellular matrix particles to be 3.0%, and adjusting the pH value of the pure water to 3.0 by hydrochloric acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.5mol/L to obtain white salting-out precipitate; soaking and dehydrating the white salting-out precipitate with ethanol for 2 hours, rinsing with PBS buffer solution, and removing residual ethanol to obtain collagen fiber; suspending collagen fibers in PBS buffer solution to make the mass percentage of the collagen fibers be 6%, and dispersing the collagen fiber suspension uniformly and fluffy by mechanical stirring; and (3) passing the collagen fiber suspension through a vacuum suction filtration device to ensure that the collagen fibers are uniformly trapped on a filter membrane with the aperture of 0.8 mu m, continuing suction filtration until the collagen fibers are in a dry semitransparent film shape, and stripping to obtain a recombinant acellular matrix layer with the thickness of 0.5 mm.
② Preparation of collagen suspension:
Firstly, preparing gel-like collagen liquid, shearing acellular matrix of submucosa of small intestine of a pig, suspending in pure water to enable the mass percentage of the acellular matrix to be 3.0%, adjusting the pH value of the pure water to 3.0 by using acetic acid, and crushing the acellular matrix to gel-like by using a tissue masher to obtain the gel-like collagen liquid;
Then preparing collagen fiber liquid, grinding the pig small intestine submucosa acellular matrix to irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5%, and adjusting the pH value of the pure water to 2.5 through acetic acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber; placing collagen fibers in acetic acid with a pH value of 3.0 to enable the mass percentage of the collagen fibers to be 1%, and mechanically stirring to enable the collagen fibers to be in a slightly-swollen and uniformly-dispersed state to obtain collagen fiber liquid;
The collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, and is prepared by mixing 30 parts of gel-like collagen liquid and 70 parts of collagen fiber liquid, uniformly stirring, and removing bubbles in vacuum.
③ Extruding and injecting the collagen suspension into a mould for forming, immersing a plurality of layers of composite pig small intestine submucosa acellular matrix layers in acetic acid with the pH of 3.0 for 30min, spreading the wetted acellular matrix on the surface of the collagen suspension, performing light pressure extrusion and air bubble generation, and standing to tightly combine the acellular matrix layers with the collagen suspension;
④ Freeze-drying the closely combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer, putting the composite freeze-dried layer into a vacuum box, vacuumizing to-110 Kpa, and keeping at 25 ℃ for 2 hours; crosslinking by gradient heating, heating to 80 ℃, and preserving heat for 3h; continuously heating to 100 ℃, and preserving heat for 3 hours; heating to 120 ℃, and preserving heat for 48 hours. And cooling to normal temperature, and taking out the collagen matrix biomembrane.
Comparative example 1
In this example, the method of example 1 was used to prepare a porcine small intestine submucosa state acellular matrix. In comparison with example 1, the collagen suspension in comparative example 1 was replaced with a collagen fibril solution.
The preparation of collagen matrix biofilm was performed as follows:
① Preparation of a dense structure acellular matrix layer: and carrying out vacuum pressing compounding on the wet acellular matrix of the 6 layers of pig small intestine submucosa to obtain a multi-layer composite acellular matrix layer.
② Preparation of collagen fiber liquid:
Preparing collagen fiber liquid, grinding the acellular matrix of the submucosa of the small intestine of the pig to irregular particles with the particle size of 0.2mm-2mm through low-temperature grinding, suspending the acellular matrix particles in pure water to enable the mass percentage of the acellular matrix particles to be 3.0%, and adjusting the pH value of the pure water to 3.0 through acetic acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber; placing collagen fibers in acetic acid with a pH value of 3.0 to enable the mass percentage of the collagen fibers to be 3%, and mechanically stirring to enable the collagen fibers to be in a slightly-swollen and uniformly-dispersed state to obtain collagen fiber liquid;
③ Extruding and injecting the collagen fiber liquid into a mould for molding, placing a plurality of layers of composite pig small intestine submucosa acellular matrix layers into acetic acid with the pH value of 3.0 for wetting for 10min, spreading the wetted acellular matrix on the surface of the collagen suspension, extruding and foaming under light pressure, and standing to tightly combine the acellular matrix layers with the collagen fiber liquid;
④ Freeze-drying the closely combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer, putting the composite freeze-dried layer into a vacuum box, vacuumizing to-110 Kpa, and keeping at 25 ℃ for 2 hours; crosslinking by gradient heating, heating to 80 ℃, and preserving heat for 3h; continuously heating to 100 ℃, and preserving heat for 3 hours; heating to 120 ℃, and preserving heat for 48 hours. And cooling to normal temperature, and taking out the collagen matrix biomembrane.
Comparative example 2
In this example, the method of example 1 was used to prepare a porcine small intestine submucosa state acellular matrix. In contrast to example 1, the collagen matrix biofilm in comparative example 2 was not cross-linked.
The preparation of collagen matrix biofilm was performed as follows:
① Preparation of a dense structure acellular matrix layer: and carrying out vacuum pressing compounding on the acellular matrix of the 6 layers of pig small intestine submucosa to obtain a multi-layer composite acellular matrix layer.
② Preparation of collagen suspension:
firstly, preparing gel-like collagen liquid, shearing acellular matrix of submucosa of small intestine of a pig, suspending in pure water to enable the mass percentage of the acellular matrix to be 1.0%, adjusting the pH value of the pure water to 3.0 by using acetic acid, and crushing the acellular matrix to gel-like by using a tissue masher to obtain the gel-like collagen liquid;
Then preparing collagen fiber liquid, grinding the pig small intestine submucosa acellular matrix to irregular particles with the particle size of 0.2mm-2mm through low-temperature grinding, suspending the particles in pure water to enable the mass percentage of the acellular matrix particles to be 3.0%, and adjusting the pH value of the pure water to 3.0 through acetic acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension; adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, centrifuging to obtain concentrated collagen fiber; placing collagen fibers in acetic acid with a pH value of 3.0 to enable the mass percentage of the collagen fibers to be 3%, and mechanically stirring to enable the collagen fibers to be in a slightly-swollen and uniformly-dispersed state to obtain collagen fiber liquid;
the collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, and is prepared by mixing 20 parts of gel-like collagen liquid and 80 parts of collagen fiber liquid, uniformly stirring, and removing bubbles in vacuum.
③ Extruding and injecting the collagen suspension into a mould for forming, placing a plurality of layers of composite pig small intestine submucosa acellular matrix layers in acetic acid with the pH of 3.0 for wetting for 10min, spreading the wetted acellular matrix on the surface of the collagen suspension, performing light pressure extrusion and bubble foaming, and standing to tightly combine the acellular matrix layers with the collagen suspension;
④ And freeze-drying the closely combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer, wherein the composite freeze-dried layer is an uncrosslinked collagen matrix biomembrane.
Comparative test
① Cytotoxicity test: the collagen matrix biofilms prepared in example 1 and comparative examples 1 and 2 were prepared according to the MTT method of the in vitro cytopenia test in the section 5 of medical device biological evaluation of national standard GB/T16886.5-2017, L929 mouse fibroblasts were selected, each group of test substances was added to a serum-containing medium, and were leached at 37 ℃ for 24 hours, and the leaching solution was added to the cells for 24 hours, and cytotoxicity detection (n=3) was performed by the MTT method, and the detection results are shown in table 1:
TABLE 1 cytotoxicity test results of collagen matrix biofilms prepared in example 1 and comparative examples 1 and 2
| Examples | Cell viability (%) |
| Example 1 | 95.60 |
| Comparative example 1 | 94.47 |
| Comparative example 2 | 95.63 |
As can be seen from Table 1, the cell viability of the sample groups of example 1 and comparative examples 1 and 2 was 93.60%, 94.47% and 95.63%, respectively. The results of cell viability in example 1 and comparative example 1 were similar, and in comparative example 2, the cell viability was slightly higher since the crosslinking treatment was not performed. Because the gradient thermal crosslinking process has very small proportion of denatured collagen fibers, the collagen matrix biomembrane after the gradient thermal crosslinking still has low cytotoxicity.
② In vitro degradability test: the samples obtained in example 1 and comparative examples 1 and 2 were weighed, each recorded as W 0, immersed in a PBS buffer containing 20U/mL collagenase at 37℃by aseptic technique, and allowed to stand. Samples were taken at 6h, 8h, and 12h, the weight measured after vacuum drying was recorded as W 1, the degradation rate was calculated according to the following formula, and the degradation calculation results are shown in Table 2:
TABLE 2 in vitro degradation detection results of collagen matrix biofilms prepared in example 1 and comparative examples 1 and 2
| Examples | Degradation rate (6 h) | Degradation rate (8 h) | Degradation rate (12 h) |
| Example 1 | 60.44％ | 78.32％ | 100％ |
| Comparative example 1 | 58.36％ | 79.92％ | 100％ |
| Comparative example 2 | 70.17％ | 90.29％ | 100％ |
The results show that example 1 and comparative example 1 have similar in vitro degradation processes, whereas comparative example 2 degrades faster as it is not crosslinked.
③ Tensile strength test: cutting the product into strip-shaped test pieces with the width of 10mm, clamping the two long ends of the product by using a clamp, starting a tensile testing machine, and testing the tensile strength (MPa) at the speed of 100 mm/min. The results are shown in Table 3:
TABLE 3 tensile Strength test results of collagen matrix biofilms prepared in example 1 and comparative examples 1 and 2
The result shows that the whole tensile strength of the collagen matrix biological film is higher, because the mechanical strength of the 6-layer composite acellular matrix is high; the acellular matrix obtained by natural tissue treatment has the phenomenon of uneven structure, so that the tensile strength of the acellular matrix is slightly deviated. The sponge layer is a relatively uniform structure, and is tested separately in order to analyze the influence of gel-like collagen liquid and gradient thermal crosslinking treatment on the mechanical properties of the final product. The results of the sponge layer showed that the collagen matrix biofilm of example 1 had higher tensile strength, the collagen matrix biofilm of comparative example 1 had inferior tensile strength, and the comparative example 2 had the lowest tensile strength. The collagen suspension of comparative example 1 uses only collagen fiber liquid without adding gel-like collagen liquid, so that the collagen fibers in the sponge layer lack entangled structure, and the tensile strength is significantly lower than that of example 1. Whereas the collagen matrix biofilm of comparative example 2 had lower tensile strength due to the uncrosslinked treatment. The results show that the collagen matrix biomembrane which is added with the gel-like collagen liquid and is subjected to gradient thermal crosslinking treatment has better mechanical strength.
④ Suture tear force test: cutting the product into strip-shaped test pieces with the width of 10mm, penetrating the product with a No. 4-0 suture line at a position 3-5 mm away from the edge of the short side, doubling the suture line, knotting the suture line at a position about 5cm away from the perforation, and preventing the suture line from falling off. And (3) respectively fixing one end of the product, which is not threaded, and one end of the suture line on a tensile testing machine, wherein the tensile speed is 100mm/min until the product is torn, and taking the maximum value of the tensile load as the tearing force (N) of the product. The results are shown in Table 4:
TABLE 4 suture tear force test results for collagen matrix biofilms prepared in example 1 and comparative examples 1 and 2
| Examples | Example 1 | Comparative example 1 | Comparative example 2 |
| Suture tear force | 6.57 | 4.63 | 4.45 |
The suture tear force test is similar to the tensile strength test result, and shows that the collagen matrix biomembrane which is added with the gel-like collagen liquid and is subjected to gradient thermal crosslinking treatment has better mechanical strength.
⑤ Wet volume stability test: appropriate amounts of the collagen matrix biofilm of example 1 and Mucograft products were cut, placed in physiological saline, and stirred at 500rpm for 3min with a magnetic stirrer to see the damage degree of the sample, and the results are shown in fig. 3, wherein a is a picture after testing of the collagen matrix biofilm of example 1 of the present application, and b is a picture after testing of Mucograft products.
The results show that the collagen matrix biofilm in example 1 can still maintain the original form after the severe stirring and breaking by the magnetic stirrer, and Mucograft products are obviously damaged and layered, so that the original form cannot be maintained, which shows that the collagen matrix biofilm in the invention obviously improves the volume stability.
While the foregoing is directed to embodiments of the present application, other and further embodiments of the application may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
Claims (13)
1. A method for preparing a collagen matrix biofilm, comprising:
s1: preparing a decellularized matrix layer with a compact structure;
S2: preparing a collagen suspension;
s3: extruding and injecting the collagen suspension into a mould for molding, then wetting the acellular matrix layer, spreading the wetted acellular matrix layer on the surface of the collagen suspension, performing light-pressure extrusion and bubble foaming, and standing to tightly combine the wetted acellular matrix layer with the collagen suspension;
s4: freeze-drying the tightly combined acellular matrix layer and the collagen suspension to obtain a composite freeze-dried layer;
S5: and (3) performing physical crosslinking on the composite freeze-dried layer to obtain the collagen matrix biomembrane.
2. The method for preparing a collagen matrix biofilm according to claim 1, wherein:
The acellular matrix layer with the compact structure is a multi-layer composite acellular matrix layer, wherein S1 further comprises the following steps,
S11, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S12 compounding: and carrying out vacuum pressing compounding on the acellular matrix of 2-6 layers to obtain a multi-layer composite acellular matrix layer.
3. The method for preparing a collagen matrix biofilm according to claim 1, wherein:
The decellularized matrix layer with a dense structure is a recombinant decellularized matrix layer, wherein S1 further comprises the steps of,
S101, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S102, crushing: crushing the acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5-3.0%, and adjusting the pH value to 2.5-3.0 by using acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension;
S103, dehydration: adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0-2.5mol/L to obtain white salting-out precipitate; soaking and dehydrating the white salting-out precipitate with an organic solvent for 0.5-2h, rinsing with PBS buffer solution, and removing the organic solvent to obtain collagen fiber;
S104, dispersing: suspending collagen fibers in PBS buffer solution to make the mass percentage of the collagen fibers be 0.1% -6%, and mechanically stirring to obtain uniformly dispersed and fluffy collagen fiber suspension;
S105, film forming: and (3) passing the collagen fiber suspension through a vacuum suction filtration device to ensure that the collagen fibers are uniformly trapped on a filter membrane with the aperture of 0.8 mu m, continuing suction filtration until the collagen fibers are in a dry semitransparent film shape, and stripping to obtain the recombinant acellular matrix layer with the thickness of 0.1-0.5 mm.
4. The method for preparing a collagen matrix biofilm according to claim 1, wherein:
The collagen suspension is prepared from gel-like collagen liquid and collagen fiber liquid, and is prepared by mixing 5-30 parts of gel-like collagen liquid and 70-95 parts of collagen fiber liquid, uniformly stirring, and removing bubbles in vacuum.
5. The method for preparing a collagen matrix biofilm according to claim 4, wherein:
The preparation method of the gel-like collagen liquid comprises the following steps:
s201 decellularization: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S202, crushing: cutting acellular matrix, suspending in pure water to make the mass percentage of acellular matrix be 0.5% -3.0%, adjusting pH to 3.0-4.0 with acid, and pulverizing acellular matrix to gel with tissue masher to obtain gel collagen liquid.
6. The method for preparing a collagen matrix biofilm according to claim 4, wherein:
the preparation method of the collagen fiber liquid comprises the following steps:
S211, cell removal: pretreating animal tissue of natural origin, inactivating virus, degreasing, decellularizing and drying to obtain a decellularized matrix;
S212, crushing: crushing the acellular matrix into irregular particles with the particle size of 0.2-2 mm through low-temperature grinding, suspending the irregular particles in pure water to enable the mass percentage of the acellular matrix particles to be 0.5-3.0%, and adjusting the pH value to 2.5-3.0 by using acid to enable the acellular matrix particles to fully swell to obtain acellular matrix suspension;
S213, dehydration: adding sodium chloride powder into the acellular matrix suspension until the concentration of sodium chloride reaches 2.0-2.5mol/L to obtain white salting-out precipitate; washing the white salting-out precipitate with PBS buffer solution, and centrifuging to obtain concentrated collagen fiber;
S214 dispersion: placing the collagen fibers in acid with the pH value of 3.0-4.0 to enable the mass percentage of the collagen fibers to be 1-6%, and mechanically stirring to enable the collagen fibers to be in a slightly-swelled uniform dispersion state to obtain collagen fiber liquid.
7. The method for preparing a collagen matrix biofilm according to any one of claims 3 or 6, wherein:
The naturally derived tissue comprises porcine or bovine small intestine submucosa, porcine or bovine peritoneum, porcine or bovine pericardium, porcine or bovine bladder, and porcine or bovine dermis.
8. A method of preparing a collagen matrix biofilm according to claim 3, wherein:
The organic solvent comprises ethanol, acetone, diethyl ether and isopropanol.
9. The method for preparing a collagen matrix biofilm according to claim 1, wherein:
The acellular matrix layer in the step S3 is subjected to wetting treatment, and is obtained by soaking the acellular matrix layer in acid with the pH value of 3.0-4.0 for 10-30 min.
10. The method for producing a collagen matrix biofilm according to any one of claims 3 or 5 or 6 or 9, wherein:
the acid comprises hydrochloric acid, acetic acid, and citric acid.
11. The method for preparing a collagen matrix biofilm according to claim 1, wherein:
The physical crosslinking in the step S5 is gradient thermal crosslinking, the composite freeze-dried layer is placed into a vacuum box, vacuumized to be less than-100 Kpa, and kept at 25-30 ℃ for 1-2 h; crosslinking by gradient heating, heating to 80-85 ℃, and preserving heat for 2-3h; continuously heating to 100-105 ℃, and preserving heat for 2-3h; heating to 120-125 deg.c and maintaining for 24-48 hr.
12. A collagen matrix biofilm characterized by:
is prepared by the preparation method of claim 1.
13. Use of a collagen matrix biofilm, comprising:
Use of a collagen matrix biofilm prepared according to the preparation method of claim 1 in any one or more of the following scenarios: the stomatology guides bone tissue regeneration, the increment of the stomatology soft tissue, prevents tissue adhesion and chronic wound repair.
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