CN118141993A

CN118141993A - Biological composite patch and preparation method and application thereof

Info

Publication number: CN118141993A
Application number: CN202410279827.4A
Authority: CN
Inventors: 吴卫东; 王佳珺; 李晓萌; 李春明; 殷敬华
Original assignee: Shanghai Weigao Medical Technology Development Co ltd
Current assignee: Shanghai Weigao Medical Technology Development Co ltd
Filing date: 2024-03-12
Publication date: 2024-06-07

Abstract

The invention provides a biological composite patch, a preparation method and application thereof, wherein the biological composite patch comprises three layers of structures which are respectively as follows: membranous acellular matrix I, acellular matrix sponge composite layer and membranous acellular matrix II; the acellular matrix sponge composite layer is embedded with a synthetic mesh. The biological composite patch provided by the invention is characterized in that the outermost acellular matrix is firstly used for directly repairing organism tissues, the tissues are regenerated for a period of time, the membranous acellular matrix and acellular matrix sponge are gradually degraded, the transmission of tissue tension is stably completed, and finally the non-absorbable material in the synthetic mesh provides long-term mechanical support, so that the repairing effect is stable for a long time. The introduction of the acellular matrix sponge significantly improves the composite effect of the acellular matrix and the synthetic mesh, and the synthetic mesh fiber monofilaments have no macroscopic pores.

Description

Biological composite patch and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a biological composite patch and a preparation method and application thereof.

Background

Soft tissue repair and reconstruction, mainly including abdominal wall defect, chest wall defect, pelvic floor muscle relaxation, etc., are an important subject of regenerative medicine. With rapid development of materialization in recent years, various repair materials such as an abdominal wall hernia patch, a hiatal hernia patch, a pelvic floor patch, etc. have been widely used in clinic.

Repair patch materials can be divided into two main categories: one type is an artificial synthetic material patch, and polymer is taken as a basic core, and the adopted polymer is polypropylene, polyester and polytetrafluoroethylene; another class is the biological patches, which are based on biological natural materials, and have been approved by the United states drug and food administration (FDA) for clinical use as materials including human dermis, porcine small intestine submucosa, porcine dermis, embryonic bovine dermis, and the like.

Synthetic materials include non-absorbable materials such as polypropylene, expanded polytetrafluoroethylene, polyester, and the like, as well as absorbable materials such as polyglycolic acid, polylactic acid glycolic acid, polylactic acid, polycaprolactone, polyvinyl alcohol, and the like. The polypropylene patch has the advantages of low cost, excellent mechanical property and stable physical and chemical properties, and is the most commonly used repairing material for abdominal wall defects at present. However, the repair process of the synthetic material is that under the chronic inflammatory stimulation of the synthetic fibers, the inflammatory cells, fibroblasts and collagen of the host proliferate into the mesh to form a composite similar to scar fiber tissue, which can cause complications such as chronic pain, patch discharge, intestinal adhesion, intestinal obstruction and even intestinal fistula. And the synthetic materials are good carriers of bacteria, and once the bacteria adhere to the synthetic materials, the bacteria form a biological film and are difficult to be destroyed by the immune system of the organism, and once serious infection occurs, the bacteria must be removed by secondary operation.

The biological patch is generally extracted from natural biological tissues through techniques such as decellularization degreasing, and the like, and can be understood as a cell scaffold, after the biological patch is implanted into a human body according to a regenerative medicine principle, a molecular biology principle and an immunology principle, the biological patch can play a good role in supporting, filling up the missing tissues of a damaged part, and under the induction of the material, the repair function of the human body can gradually grow new tissues at the original position to replace biological materials, so that the process of regenerating organ tissues is completed. Biological patches have been used in inguinal hernia repair procedures. However, because the degradation time is shorter, the degradation starts to occur in 2-4 weeks generally, the mechanical property starts to be reduced, the degradation is completed in 3-6 months, the implantation of the abdominal wall tissue is difficult to bear higher abdominal tension and compression for a long time, and the scaffold is gradually degraded after the cells grow in, so that the recurrence rate is much higher than that of the artificial patch.

To smooth the process of placing the implanted tissue-neo-tissue under tissue tension, researchers consider introducing non-degradable components or slowly degrading components. CN102266585A discloses a biological composite patch for female pelvic floor and a preparation method thereof. The patch is characterized in that the surface of a polypropylene mesh is wrapped with bladder acellular matrix, and an absorbable suture is used for suturing, so that the polypropylene mesh is wrapped. The composite patch can reduce polypropylene complications, thereby solving the problem of obvious inflammatory response at the implantation site. However, this patch still has certain problems: the suture is tied in a point-to-point manner, and the unstitched area forms a void after hydration, creating seroma that causes chronic pain.

CN102698318a discloses a preparation method of a biological material composite patch. The patch is stitched with absorbable suture using synthetic material as a base layer and absorbable material/biomaterial as an additional layer. The patch is still made of polypropylene material facing the abdominal wall layer, serious inflammatory reaction is generated at the defect part after implantation, M1 type macrophages are infiltrated by the macrophages to form a composite similar to scar fiber tissue, and the advantages of the biological material are not reflected.

CN105664257a discloses a composite soft tissue repair material with a stable repair region. The patch takes a synthetic material or a cross-linked material as a central mechanical reinforcing layer, takes a non-cross-linked membranous acellular matrix as an upper surface layer and a lower surface layer, completely covers the central mechanical reinforcing layer, and is fixed by one or more modes of medical adhesive bonding, suture bundling and vacuum lamination. Such patches combine the advantages of biological and synthetic materials, but still have certain problems: (1) For hernia patches, elasticity is required to conform to the tissue of the abdominal wall. The adhesive bonding is to fill the adhesive in the middle of the surface of the object to be bonded, and after the adhesive is cured, the surface of the object to be bonded does not generate relative movement, so that the elasticity of the patch is lost. The patch may have poor compliance of the abdominal wall during use, stress concentration is formed at the suture site, and pain and other complications are easy to occur to patients. The chitosan adhesive used in the examples of this patent also causes inflammation, and in severe cases, can cause infection, requiring a secondary surgical removal of the patch. (2) Vacuum lamination, wire binding as mentioned in the summary of the invention can avoid patch delamination, but it is practically difficult to effectively avoid this problem. Because the synthetic material and the biological material are heterogeneous materials, the vacuum lamination effect is poor, because the surface of the synthetic mesh is provided with fiber monofilament protrusions, gaps exist between the biological patch and the synthetic mesh after lamination, and the biological material and the synthetic material are easy to shift and delaminate after hydration, which is not beneficial to operation. The line binding mode is point-to-point stitching, and air bubble gaps can be formed in the unstitched area after hydration, so that serous swelling can be generated to cause chronic pain.

Current methods of compounding biological patches with synthetic mesh are generally physical bonding methods including vacuum lamination, suture tying, and medical adhesive bonding. Because synthetic mesh and biological patch are heterogeneous material, vacuum lamination effect is relatively poor to owing to synthetic mesh surface has fiber monofilament protruding, there is the space between biological patch and the synthetic mesh after the lamination, very easily takes place to shift after the hydration, leads to layering, gets into the internal acellular matrix of human body through the chamber mirror and can't be fine expansion, is unfavorable for operation. The suture tying mode is point-to-point suturing, and the unstitched area forms bubble gaps after hydration, so that seroma can be generated to cause chronic pain. Connecting the biological patch and the synthetic mesh by adopting a medical adhesive method may lose elasticity of the patch, so that the compliance of the abdominal wall of the patch is poor, and chronic pain occurs to a patient; on the other hand, new materials, adhesives, are introduced, and the biological safety and the biological compatibility need to be reevaluated.

Therefore, there is a need in the art to develop a biologic repair patch with good comprehensive properties that can induce cell ingrowth, has good mechanical strength, does not separate in the composite region after implantation, and reduces tardive pain.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a biological composite patch, a preparation method and application thereof, which not only can induce cell ingrowth, but also can have better mechanical strength, can not be separated in a composite area in early implantation, and can effectively reduce delayed pain.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a biocomposite patch comprising a three-layer structure of: membranous acellular matrix I, acellular matrix sponge composite layer and membranous acellular matrix II; the acellular matrix sponge composite layer is wrapped with a synthetic mesh.

The invention adopts the decellularized matrix sponge to wrap the synthetic mesh, the outermost membranous decellularized matrix is firstly used for directly repairing organism tissues, the growth factors in the bioactive materials are released, the fibroblast migration and proliferation are induced, and the capillary growth is induced, so that the regeneration and repair of defective tissues are realized. The tissue is regenerated for a period of time, membranous acellular matrix and acellular matrix sponge are gradually degraded, the transmission of tissue tension is stably completed, the acellular matrix is remodeled in the middle and later stages of repair, the tension-free repair of the tissue can be maintained for a long time by combining with non-absorbing permanent implantation components, and finally, the repair area achieves a long-term stable structural effect similar to that of 'reinforced concrete'. And the synthetic mesh sheet is completely wrapped in the early stage of implantation, is not directly contacted with the organism, and reduces infection caused by bacterial colonization.

Preferably, the thickness of the biocomposite patch is 0.35-0.8mm, e.g., can be 0.4mm, 0.5mm, 0.7mm, etc.

Preferably, the synthetic mesh has a thickness of 0.3 to 0.5mm, for example, 0.35mm, 0.4mm, 0.45mm, etc., a pore diameter of 2 to 6mm, for example, 3mm, 4mm, 5mm, etc., and a porosity of 50 to 90%, for example, 60%, 70%, 80%, etc.

Preferably, the synthetic mesh comprises any one of a non-absorbable mesh or a partially absorbable mesh. Wherein the non-absorbable mesh is a light mesh, and the gram weight is less than or equal to 35g/m ², for example, 34g/m²、32g/m²、30g/m²、28g/m²、26g/m²、24g/m²、22g/m²、20g/m²、18g/m²、16g/m²、14g/m²、12g/m² and the like; the gram weight of the part of the absorbable mesh after degradation, namely the gram weight of the non-absorbable part in the part of the absorbable mesh is less than or equal to 35g/m ², for example, 34g/m²、32g/m²、30g/m²、28g/m²、26g/m²、24g/m²、22g/m²、20g/m²、18g/m²、16g/m²、14g/m²、12g/m² and the like.

Preferably, the non-absorbable mesh comprises a woven from one or more of polyester, polypropylene or polyvinylidene fluoride filaments.

Preferably, the non-absorbable material in the partially absorbable mesh comprises any one or a combination of at least two of polypropylene, polyester, or polyvinylidene fluoride, and the absorbable material comprises any one or a combination of at least two of poly-4-hydroxybutyric acid, polylactic acid, polyglycolic acid, polytrimethylene carbonate, glycolide/lactide/polytrimethylene carbonate, or silk proteins.

I.e. part of the absorbable mesh is made of non-absorbable material and absorbable material.

The synthetic mesh adopted by the invention needs to meet the description of the mechanical properties of YY/T1814-2022 standard: the tensile strength is more than or equal to 16N/cm, the tearing strength is more than or equal to 16N, the sewing strength is more than or equal to 16N, and the bursting strength is more than or equal to 20kPa.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

In a second aspect, the present invention provides a method for preparing a biocomposite patch according to the first aspect, the method comprising the steps of:

(1) Grinding biological materials into powder, mixing the powder with pepsin, carrying out enzymolysis to obtain a preparation liquid, mixing the preparation liquid with a PBS solution, and regulating pH to obtain a precursor solution;

(2) Placing the precursor solution in a mould for incubation to obtain gel I, placing the synthetic mesh on the gel I, and pouring the precursor solution for incubation to form gel II, so as to obtain a gel I-synthetic mesh-gel II three-layer structure;

(3) Crosslinking the gel I-synthetic mesh-gel II three-layer structure, and freeze-drying to obtain a acellular matrix sponge composite layer;

(4) Spreading the membranous acellular matrix on a flexible substrate, placing the acellular matrix sponge composite layer, spreading the membranous acellular matrix to form a three-layer structure, and carrying out vacuum lamination compounding and drying on the three-layer structure to obtain the biological compound patch.

Preferably, the biological material is derived from any one of the mammalian hollow organ submucosa, dermis, pericardium, peritoneum, pleura or amniotic membrane.

Preferably, the mass ratio of the biological material powder to the pepsin during enzymolysis is (8-12): 1, for example, can be 9:1, 10:1, 11:1, and the like, and is preferably 10:1.

Preferably, the concentration of the biomaterial powder in the enzymolysis liquid during enzymolysis is 18-22mg/mL, for example, 19mg/mL, 20mg/mL, 21mg/mL and the like.

Preferably, the pH is adjusted to 7.2-7.4 by NaOH, for example, 7.2, 7.3, 7.4, etc.

Preferably, the concentration of the biomaterial powder in the precursor solution is 1-20mg/mL, for example, 2mg/mL, 5mg/mL, 10mg/mL, 15mg/mL, 18mg/mL, etc., preferably 5-20mg/mL.

Preferably, the mass of the gel I and the gel II and the area ratio of the synthetic mesh are respectively and independently 5-20g/100cm ², for example, 7g/100cm ²、10g/100cm²、15g/100cm²、17g/100cm²、19g/100cm² and the like.

Preferably, the ratio of the total gel mass to the synthetic mesh area in the gel I-synthetic mesh-gel II is 10-40g/100cm ², for example 15g/100cm ²、20g/100cm²、27g/100cm²、35g/100cm²、40g/100cm², etc.

Preferably, the crosslinking degree of the acellular matrix sponge in the acellular matrix sponge composite layer is 1-80%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, etc., preferably 10-48.3%.

If the crosslinking degree is low, the acellular matrix sponge is degraded firstly, and gaps still exist between the outer biological layer (namely the membranous acellular matrix) and the synthetic mesh after degradation; if the crosslinking degree is too large, the acellular matrix sponge is harder, the pore diameter becomes smaller, the synthetic mesh cannot be tightly attached during lamination, obvious gaps exist after lamination, and the lamination effect is affected. The degradation time of the acellular matrix sponge with the crosslinking degree of 10-48.3% can be effectively prolonged, and is similar to that of the outermost membranous acellular matrix.

Preferably, the vacuum lamination pressure is-0.1 to-0.092 MPa, for example, -0.098MPa, -0.096MPa, -0.094MPa, etc.

Vacuum lamination is a decellularized matrix sponge used to composite membranous decellularized matrix with a surrounding synthetic mesh. This pressure range enables the compounded patch to be hydrated and not easily separated. If the acellular matrix sponge is not contained in the biological composite patch, the composite is poor due to the following reasons: the composite effect of the acellular matrix membrane layer and the synthetic mesh is poor mainly because the mesh of the synthetic mesh is not filled with substances, and the acellular matrix membrane layer on the upper surface layer and the lower surface layer is composited only by means of vacuum lamination, so that the contact area is small, and the adhesive force is small. The acellular matrix sponge can become a softer gelatinous object after absorbing water, can be filled in meshes of the synthetic mesh, wraps the synthetic mesh, forms an integrated structure with the synthetic mesh, has larger contact area between the acellular matrix sponge composite layer and the membranous acellular matrix, has larger adhesive force, and obviously improves the effect of compositing the membranous acellular matrix and the synthetic mesh.

Preferably, the crosslinking comprises physical crosslinking or chemical crosslinking.

Preferably, the physical crosslinking includes any one of thermal crosslinking, ultraviolet crosslinking, or radiation crosslinking.

Preferably, the chemical crosslinking comprises EDC/NHS crosslinking (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-hydroxysuccinimide).

Preferably, EDC/NHS crosslinking is adopted, the EDC/NHS crosslinking does not become a part of an actual crosslinking product, but forms a water-soluble urea derivative, which can be removed by washing, so that toxic substances are prevented from being introduced, and the method has the advantages of mild reaction conditions, stable reaction products and the like.

Preferably, the EDC/NHS crosslinking comprises the steps of: preparing cross-linking liquid with EDC and NHS according to a molar ratio of 2:1, wherein the concentration of EDC in the cross-linking liquid is 30-70mM, soaking the gel I-synthetic mesh-gel II three-layer structure in the cross-linking liquid for 3-18h, taking out, cleaning the cross-linking liquid, and freeze-drying to obtain the acellular matrix sponge composite layer.

EDC/NHS crosslinking is controlled according to the parameters of the steps, so that the crosslinking degree of the acellular matrix sponge is within the range of 10-48.3%, and the acellular matrix sponge meets corresponding requirements.

In the step (4), the membranous acellular matrix is required to be paved on a flexible substrate, so that the biological material and the synthetic mesh are tightly combined, and obvious gaps do not appear around the meshes. The flexible base material is a textile material which is soft, breathable, does not deform when meeting water and does not fall off scraps, and can meet the use requirements of clean rooms, such as dust-free cloth.

In a third aspect, the present invention provides the use of a biocomposite patch as described in the first aspect for the preparation of a material for treating a hernia defect site or reducing scar formation.

The biological composite patch provided by the invention can replace the synthetic mesh in the prior art, and can be used in the aspect of the application of the synthetic mesh, such as an abdominal wall hernia patch, a hiatal hernia patch, a pelvic floor patch and the like.

Compared with the prior art, the invention has the following beneficial effects:

1. The invention adopts the decellularized matrix sponge to wrap the synthetic mesh, the outermost membranous decellularized matrix is firstly used for directly repairing organism tissues, the growth factors in the bioactive materials are released, the fibroblast migration and proliferation are induced, and the capillary growth is induced, so that the regeneration and repair of defective tissues are realized. The tissue is regenerated for a period of time, membranous acellular matrix and acellular matrix sponge are gradually degraded, the transmission of tissue tension is stably completed, the acellular matrix is remodeled in the middle and later stages of repair, the tension-free repair of the tissue can be maintained for a long time by combining with non-absorbing permanent implantation components, and finally, the repair area achieves a long-term stable structural effect similar to that of 'reinforced concrete'. And the synthetic mesh sheet is completely wrapped in the early stage of implantation, is not directly contacted with the organism, and reduces infection caused by bacterial colonization.

2. The acellular matrix sponge is subjected to light crosslinking treatment, so that the degradation time is effectively prolonged, and the acellular matrix sponge is similar to the degradation time of the outermost membranous acellular matrix, so that the generation of gaps between layers in the using process is avoided to the greatest extent.

3. The acellular matrix gel is subjected to vacuum lamination after being subjected to freeze drying treatment to form the sponge, so that the phenomena of cracking and uneven distribution of the gel in the vacuum lamination process are avoided, gaps are avoided between the acellular matrix and the synthetic mesh, and the complications such as seroma are avoided.

4. Compared with the prior art, the introduction of the acellular matrix sponge in the biological composite patch provided by the invention obviously improves the composite effect of the acellular matrix and the synthetic mesh, and the synthetic mesh fiber monofilament has no macroscopic pores and does not have layering phenomenon after hydration.

Drawings

Fig. 1 is a physical diagram of a biocomposite patch according to example 1: (a) A top view of the sample in a dry state and (b) a side view of the sample in a wet state;

FIG. 2 is a schematic structural view of a biocomposite patch according to example 1 of the present invention;

fig. 3 is a physical diagram of a biocomposite patch according to example 5: (a) A top view of the sample in a dry state and (b) a side view of the sample in a wet state;

Fig. 4 is a physical diagram of the biocomposite patch of comparative example 1 of the present invention: (a) is a side view of the sample in the dry state, (b) is a top view of the sample in the dry state, and (c) is a top view of the sample in the wet state;

Fig. 5 is a physical diagram of the biocomposite patch of comparative example 2 of the present invention: (a) A top view of the sample in the dry state and (b) a side view of the sample in the wet state.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

"Optional" or "any" means that the subsequently described event or event may or may not occur, and that the description includes both cases where the event occurs and cases where the event does not.

The indefinite articles "a" and "an" preceding an element or component of the invention are not limited to the requirement (i.e. the number of occurrences) of the element or component. Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component includes the plural reference unless the amount clearly dictates otherwise.

The description of the terms "one embodiment," "some embodiments," "exemplarily," "specific examples," or "some examples," etc., herein described means that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this document, the schematic representations of the above terms are not necessarily for the same embodiment or example.

The reagents and materials used in the examples below were obtained from conventional commercial sources without any particular explanation.

The freeze-dried non-crosslinked pig small intestine submucosa matrix (freeze-dried non-crosslinked SIS) and the freeze-dried non-crosslinked pig bladder acellular matrix (freeze-dried non-crosslinked UBM) used in the invention can be prepared by adopting the prior conventional technology or obtained from the market. The lyophilized non-crosslinked porcine small intestine submucosa matrix (SIS) used in the examples of the present invention was prepared by referring to the procedure of step 1 of example 1 in CN115006597 a; the lyophilized non-crosslinked porcine bladder acellular matrix (UBM) used in the examples of the present invention was prepared with reference to the procedure of step 1 of example 1 in CN115006597a, and the raw porcine small intestine of the procedure of step 1 of example 1 in CN115006597a was replaced with porcine bladder.

Example 1

The embodiment provides a biological composite patch, which is from bottom to top: membranous acellular matrix, acellular matrix sponge composite layer and membranous acellular matrix; the acellular matrix sponge composite layer is wrapped with a synthetic mesh;

the preparation method comprises the following steps:

(1) Crushing freeze-dried non-crosslinked SIS, mixing the obtained SIS powder with pepsin, performing enzymolysis at a pH of 4.0-4.2, wherein the concentration of the SIS powder in the enzymolysis liquid is 20mg/mL, obtaining a preparation liquid, adding NaOH into the preparation liquid, adjusting the pH to 7.2-7.4, and mixing with 0.1M PBS solution to obtain precursor solution with SIS content of 10 mg/mL;

(2) Pouring the precursor solution into a mould at the weight of 10g/100cm ², incubating for 1h at 37 ℃ to obtain gel I, placing a synthetic mesh (synthetic mesh material: polypropylene PP, aperture 2.5mm, thickness 0.43mm, gram weight 26g/m ², porosity 78%) on the gel I, pouring the precursor solution at the weight of 10g/100cm ², incubating for 1h at 37 ℃ to obtain a gel II, and obtaining a gel I-synthetic mesh-gel II three-layer structure;

(3) Preparing 50mmol/L EDC and 25mmol/L NHS crosslinking solution, soaking the gel I-synthetic mesh-gel II three-layer structure therein, taking out after soaking for 8 hours, washing the crosslinking solution, and freeze-drying to obtain the acellular matrix sponge composite layer, and sealing and preserving at 4 ℃ for later use. Determining the crosslinking degree of the acellular matrix sponge by using an ninhydrin method, wherein the crosslinking degree is 33.42%;

(4) A steel plate is taken and placed on the table top, and a piece of dust-free cloth is taken and laid on the steel plate. And (3) paving the freeze-dried non-crosslinked SIS on the non-woven fabric in a certain arrangement mode, after paving to obtain the required area and layer number, placing the acellular matrix sponge composite layer in the step (3), and paving the freeze-dried non-crosslinked SIS upwards in the same mode until the required thickness is achieved. And then sequentially placing a piece of dust-free cloth and a piece of steel plate, flattening, placing the obtained three-layer structure into a sealing bag, and carrying out vacuum lamination and compounding under the pressure of-0.096 MPa until the three-layer structure is dried, thus obtaining the biological composite patch. The thickness of the biological composite patch is 0.48mm through detection.

The physical diagram of the biological composite patch is shown in fig. 1, and as can be seen from the diagram, no macroscopic pores and bubbles are arranged beside the synthetic mesh fiber monofilaments; the structural schematic diagram of the biological composite patch is shown in fig. 2.

Example 2

The embodiment provides a biological composite patch, which is from bottom to top: membranous acellular matrix I, acellular matrix sponge composite layer and membranous acellular matrix II; the acellular matrix sponge composite layer is wrapped with a synthetic mesh;

the preparation method comprises the following steps:

(1) Crushing freeze-dried non-crosslinked UBM, mixing the obtained powder with pepsin, performing enzymolysis at a pH of 4.0-4.2, wherein the concentration of UBM powder in the enzymolysis solution is 22mg/mL, obtaining a preparation solution, adding NaOH into the preparation solution, adjusting the pH to 7.2-7.4, and mixing with 0.1M PBS solution to obtain a precursor solution with UBM content of 20 mg/mL;

(2) Placing the precursor solution in a mould according to the amount of 20g/100cm ², incubating for 1h at 37 ℃ to obtain gel I, placing a synthetic mesh (synthetic mesh material: polypropylene PP, aperture 2.5mm, thickness 0.43mm, gram weight 26g/m ², porosity 78%) on the gel I, pouring the precursor solution of 15g/100cm ², incubating for 1h at 37 ℃ to obtain gel II, and obtaining a gel I-synthetic mesh-gel II three-layer structure;

(3) Preparing cross-linking liquid with EDC and NHS according to a molar ratio of 2:1, wherein the concentration of EDC in the cross-linking liquid is 50mM, soaking the gel I-synthetic mesh-gel II three-layer structure in the cross-linking liquid for 3 hours, taking out, cleaning the cross-linking liquid, freeze-drying to obtain a acellular matrix sponge composite layer, and sealing and preserving at 4 ℃ for later use. Measuring the crosslinking degree of the acellular matrix sponge by using an ninhydrin method, wherein the crosslinking degree is 12.5%;

(4) A steel plate is taken and placed on the table top, and a piece of dust-free cloth is taken and laid on the steel plate. And (3) paving the freeze-dried UBM on the non-woven fabric in a certain arrangement mode, after paving to obtain the required area and layer number, placing the acellular matrix sponge composite layer in the step (3) in the middle, and paving the freeze-dried UBM upwards in the same mode until the required thickness is achieved. And then sequentially placing a piece of dust-free cloth and a piece of steel plate, flattening, placing the obtained three-layer structure into a sealing bag, and carrying out vacuum lamination and compounding under the pressure of-0.096 MPa until the three-layer structure is dried, thus obtaining the biological composite patch. The thickness of the biological composite patch is 0.47mm.

Example 3

the preparation method comprises the following steps:

(1) Crushing freeze-dried non-crosslinked SIS, mixing the obtained SIS powder with pepsin, performing enzymolysis at a pH of 4.0-4.2, wherein the concentration of the SIS powder in the enzymolysis liquid is 18mg/mL, obtaining a preparation liquid, adding NaOH into the preparation liquid, adjusting the pH to 7.2-7.4, and mixing with 0.1M PBS solution to obtain precursor solution with SIS content of 10 mg/mL;

(2) Placing the precursor solution into a mould in an amount of 5g/100cm ², incubating for 1h at 37 ℃ to obtain gel I, placing a synthetic mesh (synthetic mesh material: polypropylene PP, aperture 2.5mm, thickness 0.43mm, gram weight 26g/m ², porosity 78%) on the gel I, pouring 7g/100cm ² of precursor solution, incubating for 1h at 37 ℃ to obtain gel II, and obtaining a gel I-synthetic mesh-gel II three-layer structure;

(3) Preparing cross-linking liquid with EDC and NHS according to a molar ratio of 2:1, wherein the concentration of EDC in the cross-linking liquid is 50mM, soaking the gel I-synthetic mesh-gel II three-layer structure in the cross-linking liquid for 18h, taking out, cleaning the cross-linking liquid, freeze-drying to obtain a acellular matrix sponge composite layer, and sealing and preserving at 4 ℃ for later use. Measuring the crosslinking degree of the acellular matrix sponge by using an ninhydrin method, wherein the crosslinking degree is 48.3%;

(4) A steel plate is taken and placed on the table top, and a piece of dust-free cloth is taken and laid on the steel plate. And (3) paving the freeze-dried SIS on the non-woven fabric in a certain arrangement mode, after paving to obtain the required area and layer number, placing the acellular matrix sponge composite layer in the step (3), and paving the freeze-dried SIS upwards in the same mode until the required thickness is achieved. And then sequentially placing a piece of dust-free cloth and a piece of steel plate, flattening, placing the obtained three-layer structure into a sealing bag, and carrying out vacuum lamination and compounding under the pressure of-0.096 MPa until the three-layer structure is dried, thus obtaining the biological composite patch. The bio-composite patch was tested, and the thickness of the bio-composite patch was 0.49mm.

Example 4

The present example provides a biocomposite patch, which is different from example 1 only in step (3), the soaking time in step (3) is adjusted to 1h in this example, the crosslinking degree of the obtained acellular matrix sponge is 4.1%, and other raw materials, amounts and preparation methods are the same as those in example 1.

Example 5

The present example provides a biocomposite patch, which is different from example 1 only in that in step (3), the soaking time in step (3) is adjusted to 72h in this example, the crosslinking degree of the obtained acellular matrix sponge is 68.3%, and other raw materials, amounts and preparation methods are the same as in example 1.

The physical diagram of the obtained biocomposite patch is shown in fig. 3, and it can be seen from the figure that fine bubbles appear near the synthetic mesh fiber monofilaments.

Comparative example 1

This comparative example provides a biocomposite patch having the structure: acellular matrix sponge wrapped with synthetic mesh;

the preparation method was different from example 1 only in that step (4) was not performed, and other raw materials, amounts and preparation methods were the same as in example 1.

The physical diagram of the obtained biological composite patch is shown in fig. 4, and the composite scaffold is in a fluffy and porous structure, the cell-free matrix sponge is softened by water absorption after hydration, the composite patch is extremely easy to break, the composite mesh is exposed, and the composite mesh is still in direct contact with the body during actual use, so that chronic infection is extremely easy to occur.

Comparative example 2

The embodiment provides a biological composite patch, the biological composite patch includes three layer structure, three layer structure is from bottom to top respectively: membranous acellular matrix I, synthetic mesh and membranous acellular matrix II;

the preparation method comprises the following steps:

(1) The freeze-dried SIS is paved on a non-woven fabric in a certain arrangement mode, after the required area and the number of layers are paved, a synthetic mesh (the synthetic mesh material is polypropylene PP, the aperture is 2.5mm, the thickness is 0.43mm, the gram weight is 26g/m ², and the porosity is 78%) is placed, and the freeze-dried SIS is paved upwards in the same mode until the required thickness is achieved. And then sequentially placing a piece of dust-free cloth and a piece of steel plate, flattening, and carrying out vacuum lamination and compounding on the three-layer structure under the pressure of-0.096 MPa until the three-layer structure is dried, so as to obtain the biological composite patch.

The physical diagram of the biological composite patch is shown in fig. 5, and the figure shows that no acellular matrix sponge is added, obvious pores and bubbles are arranged beside the synthetic mesh fibers, and layering phenomenon is easy to occur after hydration.

Comparative example 3

This comparative example provides a biocomposite patch comprising a three-layer structure, which is from bottom to top: membranous acellular matrix I, acellular matrix sponge and membranous acellular matrix II;

the production method was different from that of example 1 only in that no synthetic mesh was added, and other raw materials, amounts and production methods were the same as those of example 1.

Test example 1

The biocomposite patches obtained in the examples and comparative examples were tested by reference to the GB/T16886 series method. Results: the cytotoxicity reactions were all grade 1; no delayed hypersensitivity; the intradermal reaction showed a difference in average score of less than 1.0 between the test sample and the solvent control; no heat generation; no hemolysis reaction; the result of the genotoxicity test shows that the salmonella typhimurium back mutation (Ames) test is a negative reaction, the mouse lymphoma test is a negative reaction, and the chromosome aberration is avoided; no acute systemic toxic reaction; no sub-chronic systemic toxicity; the tissue reaction of the muscle implanted for 30 days, 60 days and 90 days is not obviously different from that of the negative control.

Test example 2

The biocomposite patches obtained in examples and comparative examples were put into water, hydrated for 1min, surface moisture was absorbed, cut into 1×5cm specimens, and the tensile strength and elongation at break of the specimens were tested according to the method in GB/T3923.1-2013, and the results are shown in Table 1.

The biocomposite patches obtained in examples and comparative examples were observed for presence/absence of air bubbles. The biocomposite patches obtained in examples and comparative examples were cut into strips of 2cm×5cm, placed in water, hydrated for 1min, blotted to dry the surface moisture, and the samples were bent by hand to see if delamination occurred.

TABLE 1

According to the table data, when the crosslinking degree of the acellular matrix sponge is too low, the acellular matrix sponge is extremely easy to delaminate after hydration for 1min, and when the crosslinking degree is too high, tiny bubbles appear near the synthetic mesh fiber monofilaments, so that the tensile strength and the elongation at break are weakened; when the outermost membranous acellular matrix is not arranged, the acellular matrix sponge falls off after hydration, and only the synthetic mesh is left; when the acellular matrix sponge is not arranged, obvious gaps and bubbles are arranged beside the synthetic mesh fibers, and layering phenomenon is easy to occur after hydration; when the synthetic mesh is not arranged, the mechanical strength of the composite material is low, and the tensile strength cannot meet the requirement of the hernia patch.

After the hydrated layered biological composite patch enters a human body through a cavity mirror, the acellular matrix cannot be well unfolded, and the surgical operation is not facilitated. In addition, if voids such as air bubbles exist in the biocomposite patch, seroma may be generated, resulting in chronic pain.

The elongation at break in examples 1-5 can be well controlled in the range of 40-50%, and can provide good anchoring strength, reducing the problem of failure in fixation. If the elongation at break is small or basically elastic, the elastic force of the elastic force is not matched with that of fixed human tissues, so that the problem of stress concentration at the fixed position is easy to occur, and the patient is painful for a long time. If the elongation at break is too large (e.g., 90.48% of the synthetic mesh), the mesh is too soft and is prone to failure in fixation during surgery.

The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Claims

1. A biocomposite patch, characterized in that the biocomposite patch comprises a three-layer structure, the three-layer structure being respectively: membranous acellular matrix I, acellular matrix sponge composite layer and membranous acellular matrix II;

The acellular matrix sponge composite layer is wrapped with a synthetic mesh.

2. The biocomposite patch of claim 1, wherein the biocomposite patch has a thickness of 0.35-0.8mm.

3. The biocomposite patch of claim 1, wherein the synthetic mesh has a thickness of 0.3-0.5mm, a pore size of 2-6mm, and a porosity of 50-90%;

the gram weight of the non-absorbable material in the synthetic mesh is less than or equal to 35g/m ².

4. The biocomposite patch of claim 1, wherein the synthetic mesh comprises any one of a non-absorbable mesh or a partially absorbable mesh;

the non-absorbable mesh comprises one or more than one of polyester monofilament, polypropylene monofilament or polyvinylidene fluoride monofilament;

The non-absorbable material in the partially absorbable mesh comprises any one or a combination of at least two of polypropylene, polyester, or polyvinylidene fluoride, and the absorbable material comprises any one or a combination of at least two of poly-4-hydroxybutyric acid, polylactic acid, polyglycolic acid, polytrimethylene carbonate, glycolide/lactide/polytrimethylene carbonate, or silk proteins.

5. A method of preparing a biocomposite patch according to any one of claims 1 to 4, comprising the steps of:

(1) Grinding biological materials into powder, mixing the powder with pepsin, carrying out enzymolysis to obtain a preparation liquid, mixing the preparation liquid with a PBS (phosphate buffer solution) and regulating the pH value to obtain a precursor solution;

(4) Tiling the membranous acellular matrix, placing the acellular matrix sponge composite layer, tiling the membranous acellular matrix to form a three-layer structure, and carrying out vacuum lamination compounding and drying on the three-layer structure to obtain the biological compound patch.

6. The preparation method according to claim 5, wherein the mass ratio of the biological material powder to pepsin during the enzymolysis is (8-12): 1;

preferably, the concentration of the biological material powder in the enzymolysis liquid is 18-22mg/mL during enzymolysis;

preferably, naOH is adopted for adjusting the pH to 7.2-7.4;

preferably, the concentration of the biomaterial in the precursor solution is 1-20mg/mL.

7. The method of claim 5, wherein the mass to synthetic mesh area ratio of gel I, gel II is each independently 5-20g/100cm ²;

Preferably, the ratio of the total gel mass to the synthetic mesh area in the gel I-synthetic mesh-gel II is 10-40g/100cm ².

8. The method according to claim 5, wherein the acellular matrix sponge in the acellular matrix sponge composite layer has a degree of cross-linking of 1-80%, preferably 10-48.3%;

Preferably, the vacuum lamination pressure is-0.1 to-0.092 MPa.

9. The method of claim 5, wherein the crosslinking comprises physical crosslinking or chemical crosslinking;

Preferably, the physical crosslinking includes any one of thermal crosslinking, ultraviolet crosslinking, or radiation crosslinking;

Preferably, the chemical crosslinking comprises EDC/NHS crosslinking.

10. Use of a biocomposite patch according to any one of claims 1-4 for the preparation of a material for treating a hernia defect site or reducing scar formation.