CN109847099B

CN109847099B - Multilayer soft tissue repair material and preparation method thereof

Info

Publication number: CN109847099B
Application number: CN201811401674.7A
Authority: CN
Inventors: 解慧琪; 笪琳萃; 王锐; 赵龙梅
Original assignee: West China Hospital of Sichuan University
Current assignee: Ruibo Xinyuan Chengdu Biotechnology Co ltd
Priority date: 2018-11-22
Filing date: 2018-11-22
Publication date: 2022-04-01
Anticipated expiration: 2038-11-22
Also published as: CN109847099A

Abstract

The invention discloses a multilayer soft tissue repair material and a preparation method thereof, wherein the multilayer soft tissue repair material is a double-layer structure bonded by polyurethane; wherein, one layer is a small intestine submucosa membrane (SIS membrane), and the other layer is a polyurethane/small intestine submucosa composite material layer (PU/SIS composite material) formed by mixing and crosslinking polyurethane and small intestine submucosa powder. The multilayer soft tissue repair material prepared by the invention is characterized in that the SIS film and the PU/SIS composite material are firmly bonded together by the aqueous polyurethane emulsion; the material has good mechanical property and resilience, good bioactivity and biocompatibility, can be slowly degraded in vivo, overcomes the defects of the prior soft tissue repair material such as lack of elasticity and mechanical integrity, too fast in vivo degradation, low recellularization efficiency of the repair part, serious postoperative inflammatory reaction and the like, and has good application prospect.

Description

Multilayer soft tissue repair material and preparation method thereof

Technical Field

The invention relates to a multi-layer soft tissue repair material and a preparation method thereof.

Background

Soft tissue is an important tissue of the human body. However, the injury or defect of soft tissue caused by various reasons, such as disease, trauma, etc., has become one of the common clinical diseases, and seriously threatens human health. Conventional methods for treating soft tissue injuries or defects include autografting and allotransplantation, but these methods have the problems of limited donor, secondary injury, immunological rejection and the like. The method for reliably, safely and effectively repairing soft tissue injury and defect is of great significance. In recent years, with the development of tissue engineering technology, a new idea is brought to the treatment of soft tissue injury and defect by repairing and replacing damaged soft tissue by using a method of tissue engineering technology.

Small Intestinal Submucosa (SIS) is an extracellular matrix component, is rich in various growth factors, and can promote cell growth and stimulate angiogenesis under specific concentration and activity state so as to induce tissue regeneration. Moreover, SIS has the characteristics of no immunogenicity, antimicrobial activity, anisotropy and the like, and the safety and the curative effect of the SIS are verified by clinical practice. In addition, the SIS has rich and easily-obtained sources, so that the problem of limited donors in the traditional organ transplantation can be avoided, and the cost can be reduced to benefit more patients. Therefore, SIS becomes a widely researched soft tissue engineering scaffold material. Patents CN107233630A, CN107854727A and CN107281552A all refer to soft tissue repair materials obtained after SIS treatment (such as cell or extracellular matrix complex on SIS). However, like other natural biological repair materials, SIS has less than ideal mechanical properties, too fast a degradation rate and is not easy to control.

The polymer synthetic material has the advantages of excellent plastic property, controllable degradation property and mechanical property, and the like. Polyurethane (PU) is a common medical polymer synthetic material, and the combination of PU and SIS is expected to improve the mechanical property and the degradation property of a biological natural repair material, so that a more ideal soft tissue repair material is obtained. The patent CN104341608A mixes and crosslinks PU and SIS to obtain a soft tissue repair material (PU/SIS) with excellent resilience performance and mechanical property and good biocompatibility. However, the PU/SIS repair material is a single-layer repair material, and cannot well simulate and repair soft tissues with a double-layer or multi-layer structure, such as uterine wall, skin and the like.

Therefore, the finding of a multilayer material for effectively repairing soft tissues is of great significance.

Disclosure of Invention

The invention aims to provide a multilayer soft tissue repair material and a preparation method thereof.

The invention provides a multilayer soft tissue repair material, which is a double-layer structure bonded by polyurethane; wherein, one layer is a small intestine submucosa membrane, and the other layer is a polyurethane/small intestine submucosa composite material layer formed by cross-linking after mixing polyurethane and small intestine submucosa powder; the thickness ratio of the lower intestinal submucosa layer to the polyurethane/lower intestinal submucosa composite material layer is 1: 10-1: 1000; the dosage of the polyurethane emulsion is 0.02-1 mL/cm²(ii) a The polyurethane is aqueous polyurethane emulsion.

Further, the solid content of the aqueous polyurethane emulsion is 9-40 wt%.

Further, the preparation method of the intestinal submucosa membrane comprises the following steps:

taking small intestine, removing muscle layer and serosal layer, defatting, removing cells, removing scale, lyophilizing, and sterilizing;

the degreasing step comprises the steps of soaking for 10-14 hours in a mixed solution of chloroform and methanol in a volume ratio of 1:1, and washing with deionized water;

and/or the decellularization is digested by enzyme, the cells are soaked in pancreatin solution with the concentration of 0.25% overnight at the temperature of 4 ℃, and the pancreatin is removed by flushing with physiological saline;

and/or the descaling is carried out by soaking in a 0.5% sodium dodecyl sulfate solution for 4-6 h and washing with deionized water;

and/or the sterilization is sterilization using ethylene oxide.

Further, the preparation method of the polyurethane/small intestine submucosa composite material comprises the following steps:

blending the polyurethane emulsion and the powder of the lower layer of the small intestinal mucosa, introducing the suspension solution into a mold, and performing freeze-drying molding, crosslinking, washing and freeze-drying to obtain the polyurethane emulsion;

wherein the mass ratio of the polyurethane emulsion to the powder of the lower layer of the small intestinal mucosa is 5: 1-9: 1;

and/or, the freeze-drying is carried out after pre-freezing for 24 hours in a low-temperature refrigerator at the temperature of-40 ℃;

and/or the crosslinking solution is a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

and/or the crosslinking is carried out for 30-40 h at 37 ℃ in a dark reaction mode.

Further, the preparation method of the polyurethane emulsion comprises the following steps:

(1) pre-polymerization: taking a hydroxyl donor, isocyanate and a catalytic amount of catalyst for prepolymerization;

wherein the molar ratio of the hydroxyl donor to the isocyanate is 1: 0.9-1: 2; preferably, the molar ratio of the hydroxyl donor to the isocyanate is 1: 1.05-1: 1.85;

(2) chain extension: adding a chain extender for chain extension;

wherein the molar ratio of the chain extender to the hydroxyl donor in the step (1) is 1: 8-8: 1; preferably, the molar ratio of chain extender to hydroxyl donor is 1: 1;

(3) neutralization and emulsification: adding a neutralizer, uniformly stirring, dropwise adding the reaction system into a 20% (v/v) acetone aqueous solution, stirring at a high speed for 1-2 h, and removing small molecular organic matter residues to obtain the aqueous solution;

wherein, the mole ratio of the neutralizer to the chain extender in the step (2) is as follows: 0.5: 1-3: 1; preferably, the molar ratio of neutralizing agent to chain extender is 1.5: 1.

Further, in the step (1), the hydroxyl donor is any one or more of polytetrahydrofuran ether, 1, 2-pentanediol, polyethylene glycol and glycerol; preferably, the hydroxyl donor is polytetrahydrofuran ether;

and/or in the step (1), the isocyanate is any one or more of isophorone diisocyanate, methyl diisocyanate and 1, 6-hexamethylene diisocyanate; preferably, the isocyanate is isophorone diisocyanate;

and/or, in the step (1), the catalyst is stannous octoate or dibutyltin dilaurate; preferably, the catalyst is stannous octoate;

and/or, in the step (1), the prepolymerization is carried out in an oil bath kettle at 74 ℃ for 2.5-3.5 h;

and/or in the step (2), the chain extender is any one or more of 2, 2-dimethylolpropionic acid, methyldiethanolamine, dihydroxymethylpropionic acid, butanediol sulfate and sodium ethylene diamine ethanesulfonate; preferably, the chain extender is2, 2-dimethylolpropionic acid;

and/or in the step (2), the temperature of chain extension is 50-55 ℃; the chain extension time is 3-4 h;

and/or, in the step (3), the neutralizing agent is triethylamine;

and/or, in the step (3), the method for removing the small molecular organic substance residues is rotary evaporation and dialysis;

and/or in the step (3), the rotation speed of high-speed stirring is 1300 rpm.

Further, the preparation method of the small intestine submucosa powder comprises the following steps:

taking small intestine, removing muscle layer and serosal layer, defatting, removing cells, removing scale, lyophilizing, pulverizing at low temperature, and sterilizing;

and/or, after the low-temperature crushing is carried out by liquid nitrogen cooling, the low-temperature crushing is carried out by a ball mill, and the obtained product is sieved by a 200-mesh sieve;

and/or the sterilization is sterilization using ethylene oxide.

The invention also provides a method for preparing the repair material, which comprises the following steps:

coating a layer of polyurethane emulsion on the small intestine submucosa membrane, sticking the polyurethane emulsion on the polyurethane/small intestine submucosa composite material, and drying at 37 ℃ to obtain the film;

wherein the dosage of the polyurethane emulsion is 0.02-1 mL/cm²。

The invention also provides application of the multilayer soft tissue repair material in preparation of the soft tissue repair material.

Further, the soft tissue repair material is a uterine wall soft tissue repair material.

The multilayer soft tissue repair material prepared by the invention has the following beneficial effects:

(1) the invention firmly bonds the SIS film and the PU/SIS composite material together through the waterborne polyurethane emulsion; the PU/SIS composite material is used as a muscle layer and plays a supporting role on the SIS membrane layer, and the SIS membrane is used as an inner membrane layer, so that the adhesion and growth of cells are facilitated, and the repair of soft tissues can be promoted; according to the reports of the existing documents, unmodified waterborne polyurethane has poor adhesive property and is generally difficult to be used for adhesion, and the modified polyurethane material has the problems of strong toxicity and poor biocompatibility. However, the invention unexpectedly discovers that the specific SIS membrane and the PU/SIS composite material can be effectively bonded by unmodified waterborne polyurethane, so that the effective bonding is achieved, and the nontoxicity and the excellent biocompatibility are ensured.

(2) The PU/SIS composite material layer prepared by the method is of an irregular porous structure with mutually communicated holes, the porosity is high and can reach 94%, the pore diameter is uniform and ranges from 20 to 100 micrometers, and the pore diameter is suitable for cell adhesion, so that the growth of cells and capillaries is induced, and the cell proliferation is promoted;

(3) the PU/SIS composite material layer prepared by the invention has good mechanical property, has higher compressive strength and tensile strength, is close to pure PU, overcomes the defect of poor SIS mechanical strength in the prior art, and can meet the requirement of soft tissue repair materials on the mechanical property;

(4) the PU/SIS composite material layer prepared by the method has good resilience, after the 50 th cycle, the resilience and the maximum compression/tensile stress change of the PU/SIS composite material layer are close to the corresponding cycle number data of a PU group, and the requirement of a soft tissue repair material on the resilience can be still met;

(5) the SIS-PU/SIS composite scaffold prepared by the invention has good blood compatibility and histocompatibility, and can be tightly combined with peripheral cells and tissues after being implanted into in-vivo muscle tissues, so that the cells are promoted to migrate and proliferate to the interior of the material, and angiogenesis is promoted;

(6) the SIS-PU/SIS composite stent prepared by the invention is slowly degraded after being implanted into muscle tissues, and can meet the requirement of tissue growth.

In conclusion, the multilayer soft tissue repair material prepared by the invention firmly bonds the SIS film and the PU/SIS composite material together through the waterborne polyurethane emulsion; the material has good mechanical property and resilience, good bioactivity and biocompatibility, can be slowly degraded in vivo, overcomes the defects of the prior soft tissue repair material such as lack of elasticity and mechanical integrity, too fast in vivo degradation, low re-cellularization efficiency of the repair part, serious postoperative inflammatory reaction and the like, and has good application prospect.

Obviously, many modifications, substitutions, and variations may be made to the above-described embodiment without departing from the basic technical concept of the present invention in light of the above teachings and the common general technical knowledge in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a PU synthesis scheme with DMBA as the chain extender.

Of the PU of FIG. 2¹H-NMR chart.

FIG. 3 is a graph of FTIR analysis results of SIS, PU before and after crosslinking, and PU/SIS composites.

FIG. 4 is a graph showing the morphology and structure observation and pore diameter statistics of PU/SIS composite materials:

a-c: SEM results of PU/SIS1, PU/SIS2 and PU/SIS 3; d-f: graph showing HE staining results of PU/SIS1, PU/SIS2, and PU/SIS3 with glycerol mounting (scale: 100 μm); g-i: average pore size distributions of PU/SIS1, PU/SIS2, and PU/SIS 3.

FIG. 5 is a graph showing the results of compressive strength and elastic modulus of PU and PU/SIS composites, showing that the difference compared to the PU group is statistically significant, p < 0.05:

a: a compressive strength; b: modulus of elasticity.

FIG. 6 compressive cyclic stress-strain curves for PU and PU/SIS composites:

a-d: the compressive cyclic stress-strain curves for PU, PU/SIS1, PU/SIS2, and PU/SIS3, respectively.

FIG. 7 is a graph of tensile property results for PU and PU/SIS composites showing statistical differences compared to the PU group, p < 0.05:

a: tensile strength; b: modulus of elasticity; c: elongation at break; d-f: tensile cyclic stress-strain curves for PU, PU/SIS1, and PU/SIS2, respectively.

FIG. 8 is a graph showing HE dyeing results of PU and PU/SIS composites with a neutral gum blocking sheet (scale: 100 μm).

FIG. 9 is a flow chart for the preparation of an SPS composite material.

FIG. 10 is a graph showing the results of Desmin immunofluorescence staining of PP material and SPS material at 1 week of muscle implantation (scale: 50 μm).

FIG. 11 is a graph showing the results of Desmin immunofluorescence staining of PP material and SPS material at 4 weeks of muscle implantation (scale: 50 μm).

FIG. 12 is a graph showing the results of Desmin immunofluorescence staining of PP material and SPS material at 12 weeks of muscle implantation (scale: 50 μm).

Detailed Description

Abbreviation:

SIS: small intestinal submucosa, PU: polyurethane, SDS: sodium dodecyl sulfate, PTMG: polytetrahydrofuran ether, IPDI: isophorone diisocyanate, DMBA: 2, 2-dimethylolpropionic acid, TEA: triethylamine, EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, NHS: n-hydroxysuccinimide.

EXAMPLE 1 preparation of multilayer Soft tissue repair Material

1. Preparation of SIS membranes

1) Separating out small intestinal submucosa: firstly, cutting a cleaned pig small intestine into an intestine section with the length of about 15cm, scraping off a muscle layer and a serosal layer of the small intestine, washing with normal saline, and filtering to obtain a small intestine submucosa;

2) degreasing: soaking the prepared small intestine submucosa into a degreasing solution (CHCl3: CH3 OH: 1, V/V) for 12h, and repeatedly cleaning until no peculiar smell exists;

3) and (3) cell removal: immersing the degreased small intestine submucosa into a pancreatin solution with the concentration of 0.25% at 4 ℃ for overnight, rinsing until no foam exists, immersing the degreased small intestine submucosa into a lauryl sodium sulfate solution with the concentration of 0.5% for 4 hours, washing with deionized water, and freeze-drying;

4) sterilizing with ethylene oxide for later use.

2. Preparation of SIS powder

1) Separating small intestinal submucosa: cutting the cleaned small intestine of the pig into an intestine section with the length of about 15cm, scraping off a muscular layer and a serosal layer of the small intestine, washing with normal saline, and filtering to obtain a small intestine submucosa;

2) degreasing: immersing the prepared small intestine submucosa into a degreasing solution (CHCl)₃:CH₃OH 1:1, V/V) for 12 hours, and repeatedly cleaning until no peculiar smell exists;

3) and (3) cell removal: immersing the degreased small intestine submucosa into a pancreatin solution with the concentration of 0.25% at 4 ℃ for overnight, rinsing until no foam exists, immersing the degreased small intestine submucosa into an SDS solution with the concentration of 0.5% for 4 hours, washing by deionized water, and freeze-drying;

4) and (3) low-temperature crushing: and cooling the cut SIS by liquid nitrogen, crushing at low temperature by using a ball mill, sieving by using a 200-mesh sieve, and sterilizing for later use.

3. Preparation of PU emulsion

1) Pre-polymerization: PTMG 1000(33.33g, 33.33mmol), IPDI (24.18g, 96.67mmol) and stannous octoate (0.02mL) were added to a three-necked flask and reacted in an oil bath at 74 ℃ for 3 h;

2) chain extension: adding DMBA (4.94g, 33.33mmol) to react at 54 ℃ for 3h, and adjusting the viscosity of the system by using acetone if necessary in order to ensure that the reaction is smoothly carried out;

3) neutralization and emulsification: TEA (7mL) was added, stirred at room temperature for 20min, the reaction product was slowly added dropwise to an aqueous acetone solution (acetone: deionized water ═ 1:4), stirred at 1300rpm for 2h to give a PU emulsion, and small organic residues were removed by rotary evaporation and dialysis.

The PU synthesis scheme with chain extender DMBA is shown in figure 1.

Respectively subjecting the prepared PU emulsion to hydrogen nuclear magnetic resonance spectroscopy analysis¹H-MNR) and fourier infrared spectroscopy (FTIR) analysis, the results are shown in fig. 2 and 3, respectively.

As is clear from fig. 2, the synthesized PU had the highest PTMG content, and the peak areas of the nmr peaks corresponding to l and k were large at chemical shifts δ of 1.63 and 3.45ppm, respectively. Delta 2.85ppm represents the nuclear magnetic peak of j in IPDI. Delta-7.03-6.93 ppm corresponds to the nuclear magnetic peak of H on-NH-CO-. Delta-4.21 and 4.03-4.04 ppm correspond to the nuclear magnetic peaks of c and d in DMBA, respectively. Because the contents of IPDI and DMBA are lower, the nuclear magnetic resonance peak area is smaller. The triplet peak at the delta point of 1.31-1.30 ppm and the quartet peak at the 3.22-3.18 ppm are nuclear magnetic peaks of o and p in TEA respectively. No CH linked to-NCO is present at delta-3.20 ppm₂The nuclear magnetic peak corresponding to the proton above indicates that the free-NCO in IPDI has reacted to completion. The molar ratio of DMBA to PTMG to TEA in the system is 10:40:1 by calculating the nuclear magnetic peak areas at c, k and p.

As can be seen from FIG. 3, 3316cm in FTIR curve of PU^-1The absorption peak is the stretching vibration peak of N-H in the carbamate group, 2940cm^-1And 2855cm^-1Respectively corresponding to C-H stretching vibration peaks in methyl and methylene, 1698cm^-1The absorption peak at (b) is a stretching vibration peak of C ═ O in the urethane group. 1539cm^-1The position is the superposition of the C-N stretching vibration peak and the N-H bending vibration peak in the PU. The absorption peak corresponding to-NCO in IPDI is 2240-2270 cm^-1Here, the peak in the spectrum has substantially disappeared, indicating the absence of-NCO in the PU obtained, with¹The results of H-NMR were consistent.

FTIR and¹the test results of H-NMR all show that the PU emulsion is successfully synthesized in the experiment.

4. Preparation of PU/SIS composite material

1) Blending: adding 1g of SIS powder into 5g of PU emulsion with the solid content of 15 wt%, and stirring for 2.5h at room temperature to obtain a PU/SIS suspension solution;

2) freeze-drying: pouring the prepared blending system into a mould, pre-freezing in a low-temperature refrigerator at-40 ℃, and freeze-drying for 24 hours;

3) and (3) crosslinking: and soaking the freeze-dried material in EDC/NHS solution, reacting for 36h at 37 ℃ in the dark, washing for 5 times with PBS (phosphate buffer solution) for 30min each time, washing for 3 times with deionized water, and freeze-drying to obtain the PU/SIS composite material.

FTIR analysis was performed on the prepared PU/SIS composite material, and the analysis results are shown in FIG. 3.

As can be seen from FIG. 3, the FTIR curve of the crosslinked PU/SIS composite material is 1666cm^-1The absorption peak is the characteristic absorption peak of amido bond formed by EDC crosslinking of the composite material, and shows that the PU/SIS is chemically reacted under the action of the crosslinking agent EDC.

5. Preparation of SIS-PU/SIS composite material

Coating a layer of PU emulsion on the SIS film, then pasting the film on the PU/SIS composite material, drying at 37 ℃ to obtain the SIS-PU/SIS (SPS) composite material, wherein the dosage of the PU emulsion is 0.5mL/cm²。

EXAMPLE 2 preparation of multilayer Soft tissue repair Material

1. Preparation of SIS membranes

Same as in example 1.

2. Preparation of SIS powder

Same as in example 1.

3. Preparation of PU emulsion

Same as in example 1.

4. Preparation of PU/SIS composite material

1) Blending: adding 1g of SIS powder into 7g of PU emulsion with the solid content of 21 wt%, and stirring for 2.5h at room temperature to obtain a PU/SIS suspension solution;

5. Preparation of SIS-PU/SIS composite material

Same as in example 1.

EXAMPLE 3 preparation of multilayer Soft tissue repair Material

1. Preparation of SIS membranes

Same as in example 1.

2. Preparation of SIS powder

Same as in example 1.

3. Preparation of PU emulsion

Same as in example 1.

4. Preparation of PU/SIS composite material

1) Blending: adding 1g of SIS powder into 9g of PU emulsion with the solid content of 27 wt%, and stirring at room temperature for 2.5h to obtain a PU/SIS suspension solution;

As can be seen from FIG. 3, the FTIR curve of the crosslinked PU/SIS composite material is 1666cm^-1The absorption peak is the characteristic absorption peak of amido bond formed by EDC crosslinking of the composite material, and shows that PU/SIS is crosslinkedThe chemical reaction is generated under the action of EDC.

5. Preparation of SIS-PU/SIS composite material

Same as in example 1.

The beneficial effects of the invention are demonstrated in the following manner by way of experimental examples:

experimental example 1 morphological analysis of PU/SIS composite Material

1. Experimental methods

The PU/SIS composite materials PU/SIS1, PU/SIS2 and PU/SIS3 with the diameter of 6mm prepared in the embodiments 1-3 are cut into thin slices with the thickness of 300 μm, freeze-dried and sprayed with gold, and the cross-sectional structure of the materials is observed by a Scanning Electron Microscope (SEM). And HE dyeing is carried out on the PU/SIS composite material, and the appearance of the composite material is observed. And (4) carrying out statistical analysis on the pore diameter of the PU/SIS composite material by utilizing IPP software.

2. Results of the experiment

The morphology and structure observation and pore size statistics of the PU/SIS composite material are shown in FIG. 4.

As can be seen from FIG. 4, the three-dimensional structures of PU/SIS1, PU/SIS2 and PU/SIS3 are all irregular porous structures with interconnected pores. The pore diameters of the PU/SIS1, the PU/SIS2 and the PU/SIS3 are mainly distributed in the ranges of 20-120 mu m, 20-100 mu m and 20-80 mu m, and the average pore diameters are 136.89 +/-165.96 mu m, 103.82 +/-108.77 mu m and 68.85 +/-51.40 mu m respectively.

Experimental results show that the PU/SIS composite material prepared by the invention has a through three-dimensional porous structure, the aperture is uniform, the size meets the cell growth requirement, and the cell growth is facilitated.

Experimental example 2 compression Properties of PU/SIS composite Material

1. Experimental methods

The PU/SIS composite materials PU/SIS1, PU/SIS2 and PU/SIS3 prepared in the embodiments 1-3 and PU materials are taken. PU, PU/SIS1, PU/SIS2 and PU/SIS3 are prepared into a cube of 10 multiplied by 10mm by a square die, after PBS is soaked for 20min, three samples in each group are tested by a universal mechanical testing system. The experimental environment is room temperature, the testing speed is 10mm/min, the compression deformation amount of the sample is 50% of the thickness of the sample, the cycle time is 50 times, and the maximum stress and the compression modulus of the sample are recordedAnd cyclic stress-strain curves. Calculate the compression rebound resilience (R) of the sample at 10 th, 30 th and 50 th cycles_Compression) And the maximum compressive stress change of the sample at the 30 th cycle and the 50 th cycle compared to the 10 th cycle. The formula for calculating the compression resilience of the sample is as follows:

R_compression＝W_unload/W_load

W_unload: work of compression

W_load: recovery work

2. Results of the experiment

The compression properties of the PU/SIS composites are shown in FIG. 5, FIG. 6, Table 1 and Table 2.

TABLE 1 compression resilience of PU and PU/SIS composites at 10, 30 and 50 cycles.

Indicates that the difference compared to PU/SIS2 is statistically significant, p < 0.01.

TABLE 2 maximum compressive stress changes for PU and PU/SIS composites at

cycles

30 and 50 compared to cycle 10.

Indicates that the difference compared to PU/SIS2 is statistically significant, p < 0.05.

As can be seen from FIG. 5, the prepared PU/SIS composites PU/SIS1, PU/SIS2 and PU/SIS3 had compressive strengths of 47.96. + -. 3.369KPa, 151.8. + -. 6.489KPa and 270.6. + -. 22.75KPa, respectively, and elastic moduli of 213.9. + -. 14.60KPa, 967.3. + -. 192.8KPa and 1394. + -. 185.7KPa, respectively. Compared with natural SIS materials, the strength of the prepared PU/SIS composite material is obviously improved, and the compression strength and the elastic modulus of the PU/SIS2 and the PU/SIS3 are close to those of a PU group. As can be seen from FIG. 6, hysteresis loops appear in the compressive cyclic stress-strain curves for both the PU and PU/SIS composites.

As can be seen from tables 1 and 2, the PU/SIS composites prepared by the invention have good compression resilience performance, and the resilience data of the PU/SIS composites prepared by the example 2 at the 10 th cycle, the 30 th cycle and the 50 th cycle are close to that of the PU group, and the maximum compression stress at the 30 th cycle and the 50 th cycle is respectively reduced by 9.630 +/-0.35 percent and 14.36 +/-0.78 percent compared with that of the 10 th cycle, and is close to that of the PU group.

The experimental result shows that the PU/SIS composite material prepared by the invention has excellent compression strength and compression rebound resilience.

Experimental example 3 tensile Property test of PU/SIS composite Material

1. Experimental methods

The PU/SIS composite materials PU/SIS1 and PU/SIS2 prepared in the embodiments 1-2 and PU materials are taken. The PU, PU/SIS1 and PU/SIS2 were prepared as dumbbell-shaped test specimens of 75X 4X 2mm size using a dumbbell-shaped mold. After being soaked in PBS for 20min, three samples in each group are tested for tensile strength, tensile modulus and elongation at break of each sample by adopting a universal mechanical testing system. The experimental environment is room temperature, the stretching speed is 30mm/min, and the clamping length is 30 mm.

PU, PU/SIS1 and PU/SIS2 are prepared into dumbbell-shaped test samples with the size of 75 multiplied by 4 multiplied by 2mm, after being soaked in PBS for 20 minutes, three samples in each group are tested by a universal mechanical testing system. The experimental environment is room temperature, the stretching rate is 60mm/min, the clamping length is 30mm, the sample strain is 100% of the clamping length of the sample, the cycle time is 50 times, and the maximum stress and the cyclic stress-strain curve of the sample are recorded. The tensile resilience of the sample at 10 th, 30 th and 50 th cycles and the maximum change in tensile stress of the sample at 30 th and 50 th cycles compared to 10 th cycle were calculated. The number of samples was 3, and the average value was taken. The calculation formula of the tensile resilience of the sample is as follows:

R_stretching＝W_unload/W_load

W_unload: stretching work

W_load: recovery work

2. Results of the experiment

The tensile properties of the PU/SIS composites are shown in FIG. 7, Table 3 and Table 4.

TABLE 3 tensile resilience of PU and PU/SIS composites at 10, 30 and 50 cycles.

TABLE 4 maximum tensile stress change of PU and PU/SIS composites at

cycles

30 and 50 compared to cycle 10.

As can be seen from FIG. 7, the prepared PU/SIS composites PU/SIS1 and PU/SIS2 have tensile strengths of 336.7. + -. 25.17KPa and 406.7. + -. 11.55KPa, respectively, elastic moduli of 143.3. + -. 11.55KPa and 133.3. + -. 5.774KPa, respectively, and elongations at break of 452.8. + -. 11.97% and 861.4. + -. 49.69%, respectively. Although the tensile strength and elongation at break of the prepared PU/SIS composite material are lower than those of the PU group, the elastic modulus is higher than that of the PU group, and compared with the natural SIS material, the elastic modulus is obviously improved. Hysteresis loops appear on the tensile cyclic stress-strain curves of both the PU and PU/SIS composites.

As can be seen from tables 3 and 4, the PU/SIS composite materials prepared by the invention have good tensile resilience. Wherein, the rebound resilience data of the PU/SIS composite material (PU/SIS2) prepared in the example 2 at the 10 th cycle, the 30 th cycle and the 50 th cycle are 0.7281 +/-0.0069, 0.7269 +/-0.0053 and 0.7248 +/-0.0071 respectively, which are close to the rebound resilience data of the PU group at the corresponding cycle number. The maximum compressive stress of PU/SIS2 at

cycles

30 and 50 was reduced by 16.72. + -. 0.31% and 24.92. + -. 0.68% respectively, as compared to cycle 10, both close to that of the PU group.

The experimental result shows that the PU/SIS composite material prepared by the invention has excellent tensile strength and tensile resilience.

Experimental example 4 morphology analysis of PU/SIS composite Material

1. Experimental methods

And observing the distribution of the SIS in the PU/SIS composite material by using HE dyeing.

2. Results of the experiment

The HE dyeing results of PU and PU/SIS composite materials using neutral gum as an encapsulating tablet are shown in FIG. 8. Compared with PU group, the PU/SIS composite material has smaller pore diameter and more uniform pore diameter distribution. The reason for this may be that the ice crystal shape formed during prefreezing is related to the composition of the solution, the ice crystal size formed by the system is more uniform after the addition of SIS, and the pore size distribution of the lyophilized material is more uniform. Partial dissolution and discoloration of the PU occurred after contact with the neutral gum, so the PU component edge became rounded and there was evidence of dissolution observed in both the PU/SIS group and the PU group. The SIS color and morphology did not change after bonding with the neutral tree, so the SIS component appeared dark pink in the PU/SIS composite, while the PU was light pink in the system. The SIS particles are wrapped by the PU phase and are uniformly distributed in the system.

Experimental results show that the invention can successfully prepare the PU/SIS composite material with uniformly dispersed SIS.

Experimental example 5 Implantation of multilayered Soft tissue repair Material for muscle and Observation of in vivo degradation

1. Experimental methods

(1) Laboratory animal

9 healthy adult male New Zealand white rabbits with the weight of 2-3 kg are purchased from the animal culture center of Sichuan province and the ethical record number is 2017093A. The animal experiment center of Sichuan university raises for one week for later use.

(2) Experimental materials and groups

Preparing a PP and SPS composite material with the size of a block of 1cm multiplied by 0.2cm, and sterilizing the PP and SPS composite material by using ethylene oxide for later use. Before operation, the material is soaked in normal saline for 12 hours, so that the material is fully hydrated. The SPS composite material implanted group is used as an experimental group, and the PP material implanted group is used as a control group.

(3) Surgical operation

New Zealand white rabbits were fixed on their backs and anesthetized by intraperitoneal injection with 3% pentobarbital. Shaving hair on the back, fixing the back on an operation plate in a prone position, disinfecting the skin with iodophors, and paving an operation towel. Under the aseptic condition, implanting points 25-50 mm away from the spine of the New Zealand white rabbit at the back, cutting open the skin and fascia, separating the muscle gently along the long axis of muscle fiber by hemostatic forceps, implanting a reference PP and a sample SPS into the muscle with the depth of 10-20 mm, implanting the sample at one side, implanting the reference at one side, suturing the myofascium and skin incision layer by layer, and waiting for the new Zealand white rabbit to revive.

(4) General post-operative observation

Observing the survival conditions of the postoperative New Zealand white rabbits, such as diet, activity and the like, and the healing condition of wounds: such as whether the suture is dropped or not, whether the material is exposed or not, and whether the operation area has obvious weeping, pus discharge, red swelling and the like.

(5) Histological observation

At 1, 4 and 12 weeks after surgery, 3 new zealand white rabbits were randomly selected for each experimental group, and the implant material and surrounding tissues were removed. The sample is embedded by OCT frozen section embedding medium and cut by a frozen microtome. The sections were subjected to Desmin immunofluorescence staining and examined under a microscope for skeletal muscle cell migration.

The Desmin immunofluorescent staining method is as follows:

a. freezing and slicing: at 1 week, 4 weeks and 12 weeks after surgery, 3 new zealand white rabbits were randomly selected for each experimental group, and the implant material and surrounding tissues were removed. The specimen was embedded with OCT frozen section embedding medium, sliced into 6 μm thick slices with a freezing microtome, immediately fixed with 10% neutral formaldehyde at room temperature for 5min and washed with PBS.

b. And (3) sealing: adding goat serum dropwise, and sealing in a 37 deg.C incubator for 30 min.

c. Adding a primary antibody: the excess blocking solution was aspirated off the absorbent paper, and primary anti-Desmin was diluted 1:200 and added directly to the cell material complex and placed in a wet box and incubated overnight at 4 ℃.

d. Cleaning: taking out the cell material compound, re-warming for 30min at room temperature, and washing with PBS 3 times.

e. Adding a secondary antibody: absorbing excessive liquid with absorbent paper, adding freshly prepared goat anti-mouse secondary antibody (1:200) working solution dropwise onto the material, placing in a wet box, incubating at 37 deg.C for 60min, and washing with PBS for 3 times.

Dapi staining: DAPI was added dropwise and incubated for 3min in the dark, and washed with PBS.

g. Sealing: mounting the plate by using mounting liquid containing an anti-fluorescence quenching agent and collecting images under a fluorescence microscope.

2. Results of the experiment

(1) Observation of laboratory animals

All the groups of New Zealand white rabbits survived after the operation, and the animals began to eat and normally move on the day of the operation, and have no obvious change compared with the prior operation. The wound after the operation is well healed, and adverse reactions such as red swelling, infection, suture falling, graft discharging and the like are not generated.

(2) Histological observation

And (3) Desmin immunofluorescence staining observation: migration of Desmin + (skeletal muscle cells) was observed in skeletal muscle tissue of New Zealand white rabbits at different time points for PP and SPS composites. Desmin positive cells showed red color under fluorescence microscope, autofluorescence of the material showed green color at FITC channel. The nuclei and material appear blue under a fluorescent microscope. As can be seen from fig. 10, 11 and 12, the PP group was loosely connected to the surrounding skeletal muscle tissue 1 week after the operation, and no significant Desmin-positive cells were observed in the regions other than the skeletal muscle tissue. The SPS group was more tightly connected to the surrounding skeletal muscle tissue than the PP group. The SPS group observed migration and aggregation of Desmin-positive cells to the SIS layer of the SPS composite. The SPS group was more tightly connected to the surrounding skeletal muscle tissue 4 weeks post-surgery. Skeletal muscle cells began to migrate into the material in both the PP and SPS groups, with the Desmin positive cells being more abundant in the SPS composite. At 12 weeks post-surgery, the PP and SPS groups were more tightly connected to the surrounding skeletal muscle tissue than at weeks 1 and 4. Skeletal muscle cells migrate and proliferate further into the material.

The experimental result shows that the SPS composite material prepared by the experiment has good histocompatibility.

In conclusion, the multilayer soft tissue repair material (SPS composite material) prepared by the invention is characterized in that the SIS film and the PU/SIS composite material are firmly bonded together through the waterborne polyurethane emulsion; the material has good mechanical property and rebound resilience, and meets the requirement of soft tissues on the mechanical property; the porous structure can be used for cell adhesion, growth and proliferation, and can induce and promote cell growth and tissue regeneration and repair; also has good histocompatibility. Overcomes the defects of the prior soft tissue repair material such as lack of elasticity and mechanical integrity, too fast in-vivo degradation, low re-cellularization efficiency of the repaired part, heavy postoperative inflammatory reaction and the like, and has good application prospect.

Claims

1. A multi-layer soft tissue repair material, comprising: it is a double-layer structure bonded by polyurethane; wherein, one layer is a small intestine submucosa membrane, and the other layer is a polyurethane/small intestine submucosa composite material layer formed by cross-linking after mixing polyurethane and small intestine submucosa powder; the thickness ratio of the lower intestinal submucosa layer to the polyurethane/lower intestinal submucosa composite material layer is 1: 10-1: 1000; the polyurethane is aqueous polyurethane emulsion; the dosage of the polyurethane emulsion is 0.02-1 mL/cm²；

The solid content of the aqueous polyurethane emulsion is 9-21 wt%;

the preparation method of the polyurethane/small intestinal submucosa composite material comprises the following steps:

wherein the mass ratio of the polyurethane emulsion to the powder of the lower layer of the small intestinal mucosa is 7: 1-9: 1.

2. The repair material of claim 1, wherein: the preparation method of the intestinal submucosa membrane comprises the following steps:

and/or the sterilization is sterilization using ethylene oxide.

3. The repair material of claim 1, wherein:

the freeze-drying is carried out after pre-freezing for 24 hours in a low-temperature refrigerator at the temperature of-40 ℃;

4. The repair material of claim 1, wherein: the preparation method of the polyurethane emulsion comprises the following steps:

wherein the molar ratio of the hydroxyl donor to the isocyanate is 1: 0.9-1: 2;

(2) chain extension: adding a chain extender for chain extension;

wherein the molar ratio of the chain extender to the hydroxyl donor in the step (1) is 1: 8-8: 1;

wherein, the mole ratio of the neutralizer to the chain extender in the step (2) is as follows: 0.5:1 to 3: 1.

5. The repair material of claim 4, wherein: in the step (1), the molar ratio of the hydroxyl donor to the isocyanate is 1: 1.05-1: 1.85.

6. The repair material of claim 4, wherein: in the step (2), the molar ratio of the chain extender to the hydroxyl donor is 1:1.

7. The repair material of claim 4, wherein: in the step (3), the molar ratio of the neutralizing agent to the chain extender is 1.5: 1.

8. The repair material of claim 4, wherein:

in the step (1), the hydroxyl donor is one or more of polytetrahydrofuran ether, 1, 2-pentanediol, polyethylene glycol and glycerol;

and/or in the step (1), the isocyanate is any one or more of isophorone diisocyanate, methyl diisocyanate and 1, 6-hexamethylene diisocyanate;

and/or, in the step (1), the catalyst is stannous octoate or dibutyltin dilaurate;

and/or in the step (2), the chain extender is any one or more of 2, 2-dimethylolpropionic acid, methyldiethanolamine, dihydroxymethylpropionic acid, butanediol sulfate and sodium ethylene diamine ethanesulfonate;

and/or, in the step (3), the neutralizing agent is triethylamine;

and/or in the step (3), the rotation speed of high-speed stirring is 1300 rpm.

9. The repair material of claim 8, wherein:

in the step (1), the hydroxyl donor is polytetrahydrofuran ether;

and/or, in the step (1), the isocyanate is isophorone diisocyanate;

and/or, in the step (1), the catalyst is stannous octoate;

and/or, in the step (2), the chain extender is2, 2-dimethylolpropionic acid.

10. The repair material of claim 1, wherein: the preparation method of the small intestine submucosa powder comprises the following steps:

and/or the sterilization is sterilization using ethylene oxide.

11. A method of producing the repair material according to any one of claims 1 to 10, characterized in that: the preparation method comprises the following steps:

wherein the dosage of the polyurethane emulsion is 0.02-1 mL/cm²。

12. Use of the multilayer soft tissue repair material according to any one of claims 1 to 10 for the preparation of a soft tissue repair material.

13. Use according to claim 12, characterized in that: the soft tissue repair material is a uterine wall soft tissue repair material.