CN114432501A

CN114432501A - Rotator cuff patch and preparation method thereof

Info

Publication number: CN114432501A
Application number: CN202210114561.9A
Authority: CN
Inventors: 何红兵; 尹荣鑫; 周星宇; 杨莉; 苏岭; 闫侃
Original assignee: Shanghai P & P Biotech Co ltd
Current assignee: Shanghai P & P Biotech Co ltd
Priority date: 2022-01-30
Filing date: 2022-01-30
Publication date: 2022-05-06
Also published as: WO2023143337A1

Abstract

The invention provides a rotator cuff patch and a preparation method thereof. The rotator cuff patch has a three-dimensional nano-mesh structure and is made of raw materials containing a fibrinogen complex and polylactic acid-caprolactone. The rotator cuff patch is prepared by adding a fibrinogen composite solution and a polylactic acid-caprolactone solution into the same volumetric tube of an electrostatic spinning machine or respectively adding the fibrinogen composite solution and the polylactic acid-caprolactone solution into different volumetric tubes of the electrostatic spinning machine, performing electrostatic spinning, and drying. The rotator cuff patch provided by the invention has a nano-scale three-dimensional structure similar to extracellular matrix and good hydrophilicity; the self tendon can be formed in the shoulder joint cavity after the implantation for about 6 weeks, and the self tendon and the shoulder joint bone tissue form tendon bone healing; the maximum force of the regenerated rotator cuff tissue is restored to 95-100% of the normal side about 24 weeks after implantation. The rotator cuff patch can be used for bridging or strengthening treatment of large and ultra-large rotator cuff tissue lacerations.

Description

Rotator cuff patch and preparation method thereof

Technical Field

The invention belongs to the technical field of medical instruments, and particularly relates to a rotator cuff patch and a preparation method thereof.

Background

The stability and function of the shoulder joint is largely dependent on the integrity of the rotator cuff muscle group. Acute rotator cuff tears are common in athletes, chronic degenerative tendon changes and subsequent tears are common in the elderly and in sedentary patients. The frequency and magnitude of rotator cuff tears increases with age. Rotator cuff injuries do not readily heal on their own, which is associated with complex anatomy of the shoulder joint, large range of motion, relative weakening of the tendons, and low vascularization. Untreated partial rotator cuff tears, whether traumatic or degenerative, develop into large and extra-large tears. Manifested by local pain and suppression of the muscle motor reflex, reduced motor ability and even endangering daily activities. As the condition progresses, the stability and mechanical properties of the shoulder joint may be further compromised, eventually leading to irreversible complete rotator cuff tear arthropathy syndrome.

Currently, the high recurrence rate after rotator cuff repair is a significant challenge for surgeons. Small (<1cm) to medium (1-3cm) lacerations with a recurrence rate of about 26%; the large (3-5cm) and large (more than 5cm) type tears, the recurrence rate can reach 94%.

Methods of treating rotator cuff tears include various suturing techniques (e.g., single and double row suture anchors), tissue transplantation (allograft, xenograft, autograft, acellular matrix), and tissue regeneration. The normal tendon healing process is divided into three mutually overlapping stages, including 1) an inflammation stage, within 4-7 days after the operation, collagen deposition and inflammatory cell migration to the recovery site; 2) fibroblast and tendon cell proliferation and repair to form an extracellular matrix consisting of temporary collagen; 3) after 6 to 8 weeks, tendon tissue composed of collagen arranged in an orderly manner is formed.

At present, rotator cuff tear repair is mainly to fix tendons to bone ends by using a suture anchor, and the limitations can be summarized into three types: (1) the normal healing process of the tendon cannot be reproduced, and the tissues formed after repair mainly consist of fibrovascular scar tissues. (2) Four areas where the tendon bone heals eventually cannot be reproduced at the interface where the tendon is attached to the bone tissue (start and stop points). The start and stop point is a complex biological connection that allows mechanical stress to be transferred between different materials. The mechanical property of the fibrovascular scar tissue formed on the repaired interface is different from that of a normal tendon structure, so that the repaired part is easy to break down. (3) Sufficient mechanical strength cannot be obtained after the repair.

Clinically, neither the commonly used suturing techniques nor tissue transplantation are satisfactory for the treatment of large and ultra-large rotator cuff tears, thus prompting the development of tissue regeneration techniques. The tissue regeneration technology mainly comprises the steps of adopting cells, growth factors and biomaterial matrixes, and singly or in combination simulating the fibrous structure of the extracellular matrix so as to finally recover the mechanical and physiological properties of the rotator cuff tendon. In orthopedic practice, the clinical treatment of large or giant rotator cuff defects is often less effective than small or medium tears. Related research has focused on alternative reconstruction techniques for large or ultra-large tears. Various technical strategies have been proposed, and patch patching is one of the popular research topics among them.

Sano et al used autologous fascia transplantation to repair supraspinatus defects in rabbits. During survival cell density gradually increased and the patch was histologically similar to normal tendon structure at 8 weeks post-surgery. The problem with this study is that the use of autografts can cause secondary damage to the donor site.

Synthetic grafts have been used with some success for rotator cuff tear repair, with the greatest advantage of avoiding secondary injury to the donor site. Polytetrafluoroethylene (PTFE) was used to reconstruct irreparable rotator cuff tears with inconsistent clinical results. Some studies report good clinical results with pain relief and also have found that bone resorption occurs at the native tissue-graft bone attachment. Kimura et al used PTFE as a graft to repair the irreparable infraspinatus tear in a canine model and found that the snap-off test failed within PTFE immediately after implantation. After 12 weeks, all animals failed at the PTFE-bone interface.

Absorbable biological patches (e.g., allograft, xenograft) are also used in clinical practice. Porcine Small Intestinal Submucosa (SIS) is a biomaterial that can act as a scaffold for tendon, ligament, and fascia reconstruction with minimal host response. Dejardin et al induced repair of rotator cuff tears in a canine model using SIS. After 3 months and 6 months of operation, the patch-tendon tissue becomes thinner obviously, and is similar to the contralateral tendon tissue, and the bone-patch-tendon tissue has continuity. Fibroplasia and collagen deposition were found in sham surgery and SIS specimens 3 months post-operatively. Regeneration structures close to normal tendons can be observed within 6 months. The maximum force at all time points was much less for the sham and SIS patches compared to normal infraspinatus (16.57% -49.73%).

Julie E reports that the results of the dog animal test using human dermal acellular matrix showed that, although the maximum forces at 3 months (538.6N) and 6 months (552.4N) after surgery were 8-9 times higher than immediately after surgery (65.5N), only 58-60% of the normal control (918.7N) was recovered.

The existing patch has no ideal effect on repairing rotator cuff tear, so that an implant with better rotator cuff tear treatment effect still needs to be researched.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a rotator cuff patch and a preparation method thereof. The rotator cuff patch has a nanoscale three-dimensional structure similar to extracellular matrix and good hydrophilicity, can form tendons in a shoulder joint cavity after being implanted for about 6 weeks, and can form tendon bone healing with shoulder joint bone tissues; the maximum force of the regenerated rotator cuff tissue is recovered to 95-100% of the normal side about 24 weeks after the implantation; the rotator cuff patch can be used for bridging or strengthening treatment of large and ultra-large rotator cuff tissue lacerations.

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

in a first aspect, the invention provides an application of a hydrophilic electrostatic spinning biological composite scaffold material in preparing an implant for treating rotator cuff tissue tearing, wherein the composite scaffold material is prepared by blending an aqueous solution of fibrinogen, L-arginine or hydrochloride thereof and a P (LLA-CL) solution and adopting an electrostatic spinning technology; wherein the mass ratio of the fibrinogen to the L-arginine or the hydrochloride thereof is 1.2: 1-12.5: 1;

the fibrinogen and L-arginine or hydrochloride aqueous solution thereof, wherein the solvent is selected from one or more of pure water, water for injection, salt solution and buffer solution; the salt solution is selected from sodium chloride solution and potassium chloride solution; the buffer solution is selected from phosphate buffer solution, Tris-HCl buffer solution, glycine buffer solution and D-Hank's solution.

In some embodiments of the invention, the fibrinogen is mammalian-derived fibrinogen.

In some embodiments of the invention, the mammal is a human, pig, cow, sheep or horse.

In some embodiments of the invention, the mass ratio of the polylactic acid to the polycaprolactone in the P (LLA-CL) is 20:80 to 95: 5.

In some embodiments of the present invention, the solvent in the P (LLA-CL) solution is selected from one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran, chloroform, or acetone.

In some embodiments of the invention, the fibrinogen, L-arginine or hydrochloride aqueous solution is blended with the P (LLA-CL) solution, wherein the mass ratio of fibrinogen to P (LLA-CL) is 0.2:1 to 2.1: 1.

In some embodiments of the invention, the hydrophilic electrospun biocomposite scaffold material has an equilibrium contact angle of less than 55 °.

In some embodiments of the invention, the hydrophilic electrospun biocomposite scaffold material has a total volume shrinkage of no greater than 20% after contacting an aqueous solution; the porosity is not less than 30%.

In some embodiments of the present invention, the aqueous solution of fibrinogen, L-arginine or hydrochloride thereof is further loaded with an antibacterial substance selected from one or more of penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, macrolides, glycosides, sulfonamides, quinolones, nitroimidazoles, lincomamines, fosfomycin, chloramphenicol, para-colistin B, bacitracin.

In some embodiments of the invention, the penicillin is selected from the group consisting of penicillin, ampicillin, carbenicillin; the cephalosporins are selected from cefalexin, cefuroxime sodium, ceftriaxone and cefpirome; the carbapenem is thiomycin; the aminoglycoside is selected from gentamicin, streptomycin, and kanamycin; the tetracycline is selected from tetracycline and chlortetracycline; the macrolides are selected from erythromycin and azithromycin; the glucoside is vancomycin; the sulfanilamide is selected from sulfadiazine and trimethoprim; the quinolone is selected from the group consisting of pipemidic acid and ciprofloxacin; the nitroimidazoles are selected from metronidazole and tinidazole; the lincomycin is selected from lincomycin and clindamycin.

In some embodiments of the invention, the amount of the antimicrobial substance released is no less than 30% of the total loading within 15 minutes after implantation of the scaffold material in vivo.

The invention provides a rotator cuff patch which has a three-dimensional nano-net structure and is prepared from raw materials comprising a fibrinogen compound and polylactic acid-caprolactone.

In some embodiments of the present invention, the fibrinogen complex comprises the following components in parts by weight:

fibrinogen 0.1-20 parts (for example, may be 0.1 part, 0.5 part, 1 part, 1.5 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 12 parts, 13 parts, 15 parts, 18 parts or 20 parts, etc.), arginine hydrochloride 0.1-10 parts (for example, may be 0.1 part, 0.5 part, 0.8 part, 1 part, 1.2 parts, 1.5 parts, 1.8 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts, etc.), sodium chloride 0.01-10 parts (for example, may be 0.01 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 parts, 1 part, 2 parts, 3 parts, 5 parts, 1.8 parts, 2 parts, 1.8 parts, 2 parts, 1.8 parts, 2 parts, 1.8 parts, 1, 2 parts of sodium citrate, 2 parts, 1.8 parts, 2 parts of sodium citrate, 1, 2 parts, 1.8 parts, 2 parts of sodium citrate, 1.8 parts, 2 parts, 1.8 parts, 2 parts, 1.8 parts of sodium citrate, 2 parts, 1.8 parts, 2 parts of sodium citrate, 2 parts, 1.8 parts, 1, 2 parts of sodium or 10 parts of sodium citrate, etc.), etc., sodium citrate, etc., sodium or 2 parts, etc, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc.).

Preferably, the fibrinogen complex comprises the following components in parts by weight:

3-15 parts of fibrinogen, 0.5-5 parts of arginine hydrochloride, 0.3-5 parts of sodium chloride and 1-10 parts of sodium citrate.

In the present invention, the source of the fibrinogen is not particularly limited, and may be, for example, a source derived from a mammal such as a human, pig, cow, sheep, horse, etc., and generally, if not specifically mentioned, a fibrinogen derived from pig blood is preferable, and the fibrinogen complex contains a coagulable protein as a main component, and various excipients or protective agents.

In some embodiments of the invention, the polylactic acid-caprolactone has Mn of 30000-80000 (e.g., 30000, 40000, 50000, 60000, 70000, or 80000, etc.), Mw of 80000-160000 (e.g., 80000, 90000, 100000, 110000, 120000, 130000, 140000, 150000, or 160000, etc.), and Mw/Mn <3.0 (e.g., 2.9, 2.6, 2.5, 2.2, 2.0, 1.8, or 1.5, etc.).

In some embodiments of the invention, the mass ratio of the polylactic acid chain segment to the polycaprolactone chain segment in the polylactic acid-caprolactone is 20: 80-95: 5; for example, it may be 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, or 95:5, etc.

In some embodiments of the invention, the rotator cuff patch has a thickness of 1.6-10 mm, for example, 1.6mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, or 10 mm; preferably 2 to 3 mm.

In some embodiments of the invention, the protein content in the rotator cuff patch is 100-220mg/g, such as 100mg/g, 120mg/g, 130mg/g, 150mg/g, 160mg/g, 180mg/g, 200mg/g or 220 mg/g; the residual protein content is <12mg/g, and may be, for example, 11mg/g, 10mg/g, 9mg/g, 8mg/g, 7mg/g, 6mg/g, 5mg/g, 3mg/g, 2mg/g, 1mg/g, or the like.

In some embodiments of the invention, the water extract of the rotator cuff patch has a pH of 6 to 8.

In some embodiments of the invention, the rotator cuff patch has a porosity of 41-100%, for example 41%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; preferably 41 to 80%.

In some embodiments of the present invention, the rotator cuff patch has a water absorption of 35 to 200%, for example, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 120%, 130%, 150%, 160%, 180%, or 200%, etc.; preferably 55 to 80%.

In some embodiments of the invention, the rotator cuff patch has a tensile strength of 2 to 5MPa, for example, 2MPa, 2.5MPa, 3MPa, 3.5MPa, 4MPa, 4.5MPa, or 5 MPa.

In some embodiments of the invention, the rotator cuff patch has an elongation at break of 100 to 200%; for example, it may be 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, etc.

In some embodiments of the invention, the rotator cuff patch further comprises a drug having an antibacterial and/or anti-inflammatory effect.

In a third aspect, the present invention provides a method for preparing a rotator cuff patch according to the second aspect, comprising the steps of:

mixing the fibrinogen compound solution and the polylactic acid-caprolactone solution, adding the mixture into the same volumetric tube of an electrostatic spinning machine, and performing electrostatic spinning; or adding the fibrinogen composite solution and the polylactic acid-caprolactone solution into different volumetric tubes of an electrostatic spinning machine respectively, and performing electrostatic spinning simultaneously; and drying to obtain the rotator cuff patch.

In the present invention, fibrinogen is denatured after electrostatic spinning, and the denatured fibrinogen is a poorly water-soluble protein which retains only the primary structure (amino acid sequence) of fibrinogen and loses the secondary and tertiary and quaternary structures. The proteins contained in the rotator cuff patch of the present invention are mainly the denatured fibrinogen, and may have a small amount of undenatured water-soluble fibrinogen. The residual protein in the invention is the water-soluble fibrinogen.

In some embodiments of the invention, the concentration of the polylactic acid-caprolactone solution is 5-8 g/100 mL; for example, it may be 5g/100mL, 5.5g/100mL, 6g/100mL, 6.5g/100mL, 7g/100mL, 7.5g/100mL, 8g/100mL or the like.

In some embodiments of the present invention, the solvent of the polylactic acid-caprolactone solution is selected from one or a combination of at least two of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran and acetone. In the present invention, the amount of the organic solvent remaining in the rotator cuff patch is preferably controlled to be less than 0.55 mg/g.

In some embodiments of the invention, the solvent of the fibrinogen complex solution is water or an aqueous solution.

In some embodiments of the invention, the concentration of the fibrinogen complex solution is 8-29g/100 mL; for example, the concentration may be 8g/100mL, 9g/100mL, 10g/100mL, 12g/100mL, 15g/100mL, 18g/100mL, 20g/100mL, 22g/100mL, 25g/100mL, 27g/100mL, 29g/100mL, or the like.

In some embodiments of the invention, the mass ratio of the fibrinogen complex to the polylactic acid-caprolactone in the fibrinogen complex solution and the polylactic acid-caprolactone solution is 0.48-1.1: 1; for example, it may be 0.48:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, or 1.1: 1.

In some embodiments of the invention, the electrospinning conditions are: the voltage difference is 15-140kV (for example, 15kV, 20kV, 30kV, 40kV, 50kV, 60kV, 70kV, 80kV, 90kV, 100kV, 110kV, 120kV, 130kV or 140kV, etc.), the solution flow rate is 3-399mL/h or 401-960mL/h (for example, 3mL/h, 10mL/h, 20mL/h, 50mL/h, 80mL/h, 100mL/h, 200mL/h, 300mL/h, 399mL/h, 401mL/h, 500mL/h, 600mL/h, 700mL/h, 800mL/h, 900mL/h and 960mL/h, etc.), and the distance between the needle and the mandrel is 10-50cm (for example, 10cm, 20cm, 30cm, 40cm or 50cm, etc.).

In some embodiments of the invention, the method of preparing further comprises: and the surface of the rotator cuff patch is coated with a medicament with antibacterial and/or anti-inflammatory effects.

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

(1) the rotator cuff patch provided by the invention has a three-dimensional nano-mesh structure, the protein content of the rotator cuff patch is 100-220mg/g, the residual protein content is less than 12mg/g, the porosity is 41-100%, the water absorption is 35-200%, the tensile strength is 2-5 MPa, the elongation at break is 100-200%, and the pH value of a water extract is 6-8, so that the rotator cuff patch has good hydrophilicity and tensile strength. The rotator cuff patch can regenerate tendon tissues in a joint cavity after being implanted for about 6 weeks, and forms tendon bone healing at a joint part of bones and tendons so as to replace the injured tendons to play a role.

(2) The rotator cuff patch provided by the invention can be completely degraded in an in-vivo environment, and simultaneously induces rotator cuff tissue regeneration in situ. Through animal experiment results, we surprisingly found that the maximum breaking force and tensile strength of the regenerated tendon gradually increase along with the in vivo implantation time of the rotator cuff patch, and the maximum breaking force of the regenerated tendon reaches the level of a normal control group (> 95%) and is far greater than that of an SIS material (16.57% -49.73%) reported by Dejardin and a human dermal acellular matrix (58-60%) reported by Julie E24 weeks after operation.

(3) The rotator cuff patch provided by the invention is prepared from a synthetic absorbable polymer and a natural high molecular biological material, does not need to adopt any chemical or biological cross-linking agent for cross-linking and fixing, does not contain bioactive factors and living cells, and omits any tissue culture or tissue preparation process, thereby reducing the cost and logistics complexity. In addition, the implant material is manufactured by adopting the electrostatic spinning technology, so that the quality parameters of the product can be effectively designed and controlled by controlling raw materials (such as composition and proportion), equipment parameters (such as voltage, concentration, distance and the like) and the like, and the characteristics (such as mechanical strength and degradation rate), induced functionality, remodeling regeneration rate and the like of the implant are adjusted, so that the mass production and the popularization and application of innovative products are facilitated.

Drawings

FIG. 1 is a schematic view showing the shape of a rotator cuff patch used in an animal experiment according to the present invention;

FIG. 2 is a schematic view of the sewing mode of the shoulder sleeve patch in the animal experiment;

wherein A, B are opposite sides of the rotator cuff patch.

FIG. 3 is a sampling site diagram in the animal experiment of the present invention.

Fig. 4 is a cross-sectional view of a sample humerus on the surgical side taken at various observation times after scheduled surgery.

FIG. 5 is a graph comparing the maximum force to break of the surgical side and normal side samples taken at predetermined post-operative observation times.

FIG. 6 is a graph showing a comparison of tensile strengths of the samples of the operation side and the normal side taken at predetermined observation times after the operation.

FIG. 7 is an optical micrograph of HE staining and Masson staining of regenerated tendon tissue in the joint cavity at each observation time after the scheduled surgery.

FIG. 8 is an HE stained optical micrograph of regenerated tendon bone healing structures at various observation times after a predetermined period of time;

wherein, 6 weeks after surgery: a (× 20) and D (× 40); 12 weeks after surgery: b (× 20) and E (× 40); 24 weeks after surgery: c (. times.20) and F (. times.40).

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the embodiment of the invention, the adopted part of raw materials are as follows:

polylactic acid-caprolactone (PLCL): mn of 50000, Mw of 100000, Mw/Mn of 2.0, and the mass ratio of the polylactic acid segment to the polycaprolactone segment of 70:30, which is purchased from Purac, Netherlands;

fibrinogen: common porcine fibrinogen.

Example 1

The embodiment provides a rotator cuff patch, which is prepared by the following steps:

(1) dissolving PLCL in hexafluoroisopropanol to form a PLCL solution with a concentration of 8g/100 mL;

dissolving a fibrinogen complex (hereinafter referred to as F-Fg) in distilled water to form an F-Fg solution having the following component concentrations: 12.61g/100mL of fibrinogen (Fg), 3.29g/100mL of arginine hydrochloride, 3.27g/100mL of sodium chloride and 9.83g/100mL of sodium citrate;

(2) stirring and mixing the PLCL solution and the F-Fg solution for 5 hours according to the volume ratio of 0.77:0.23 to form a mixed electrostatic spinning solution, wherein the mass ratio of the F-Fg to the PLCL is 1.08: 1;

(3) transferring the mixed solution into a liquid carrying device of a mass production electrostatic spinning machine (model: NS1WS 500Elmarco Czech) to carry out electrostatic spinning, wherein the process conditions are as follows: the voltage difference is 100kV, the flow rate of the electrostatic spinning solution is 60mL/h, and the distance between a spinning electrode and a base material is 14 cm; the prepared electrostatic spinning film is dried for 48 hours in vacuum at room temperature, and the rotator cuff patch with the three-dimensional net structure is obtained, wherein the thickness of the rotator cuff patch is 1.6 mm.

Examples 2 to 5

Examples 2-5 each provide a rotator cuff patch which was prepared by a method different from that of example 1 only in the concentration of F-Fg in the mixed electrospinning solution and the mass ratio of F-Fg to PLCL, as shown in table 1 below.

TABLE 1

Physical characterization

The physical parameters of the rotator cuff patch obtained in the above embodiment are characterized and tested by the following method:

1. tensile strength and elongation at break

The test was performed using a high temperature mechanical tester (Instron-5567) with a gauge length of 20mm and a crosshead speed of 20 mm/min. Samples of 1.5cm x 7cm in size and 0.2 to 0.4mm in thickness were prepared and the test was carried out at room temperature at a relative humidity of 60% according to the method of the above examples and comparative examples, with 5 readings per set of samples and averaged.

2. Porosity of the material

Porosity: the porosity (P) was tested by the impregnation method. Soaking the sample in absolute ethyl alcohol for 12 hours, measuring the mass of the sample before and after soaking, and calculating the porosity according to the following formula:

wherein W is the mass of the soaked sample, W₀Rho is the density of absolute ethyl alcohol, and V is the apparent volume of the sample.

3. Water absorption rate

The principle is as follows: the sample was immersed in distilled water at 23.0. + -. 1.0 ℃ for a certain period of time, and the difference in mass between the time of starting the test and the time after water absorption of the sample was measured and expressed as the percentage of the difference in mass with respect to the initial mass.

Preparation of the sample: 3 samples with a mass of about 0.5 g/piece were cut out for measurement.

And (3) determination of a sample: 1) the samples were placed in a weighing bottle, placed in an oven at 50.0 ℃ ± 2.0 ℃ for at least 24h, then cooled to room temperature in a desiccator, each sample was weighed to the nearest 0.1mg, and the procedure was repeated until the mass change of the sample was within ± 1 mg.

2) The sample was placed in a container with distilled water, the water temperature being controlled at 23.0 ℃. + -. 1.0 ℃.

3) After soaking for 1 plus or minus 0.1h, taking out the samples, gently wiping off water on the surfaces of the samples by using a clean dry cloth or filter paper, and weighing each sample again to be accurate to 0.1 mg. After the sample is taken out of the water, weighing should be completed within 1 min.

The water absorption of the sample was calculated using the formula:

c(％)＝(m₂-m₁)/m₁×100％；

in the formula: c, the water absorption mass fraction of the sample, wherein the numerical value is expressed in percent;

m₂the mass of the soaked sample is mg;

m₁the mass of the sample before and after drying before soaking is in mg.

4. Protein content

The principle is as follows: the protein molecule contains more than two peptide bonds, and can be combined with Cu in alkaline solution²⁺Forming a purple red complex, wherein the color intensity is in direct proportion to the protein concentration in a certain range, taking a protein reference substance solution as a standard curve, and determining the content (mg/mL) of protein in a test sample by a colorimetric method, and converting the content into the content (mg/g) of protein in a sample.

And (3) determination of a sample: the sample is cut into pieces, 100mg is accurately weighed, and the pieces are put into a sample tube (16 pieces of 3mm and 2 pieces of 5mm magnetic beads are arranged inside) attached to a biological sample homogenizer, and 3mL of biuret test solution is added. Setting a homogenizing speed: 6m/sec, time: samples were prepared as suspensions 20sec × 5 times, with temperature control below 37 ℃. Centrifuging at 4000rpm for 15min until precipitate is removed, collecting supernatant as test solution, and using within 120 min. Parallel samples were prepared in the same manner.

Precisely transferring 1mL of the test solution into 3 test tubes (parallel samples), adding 4mL of biuret test solution, mixing, standing at room temperature for 30min, and determining absorbance at 540nm wavelength according to 0401 ultraviolet-visible spectrophotometer method in the fourth part of the pharmacopoeia of China (2015 edition); meanwhile, physiological saline is used as a blank sample.

And (4) calculating a result:

calculating the protein concentration of the test solution according to the absorbance and the standard curve, and converting the protein content (mg/g) in the sample according to the following formula:

wherein, C is the protein content (mg/g) in the sample;

C_tsthe final concentration of protein in the test solution (mg/mL);

w: weight of sample in sample tube (mg) attached to each tube of biological sample homogenizer;

v: volume (mL) of biuret test solution added to the sample tube attached to the biological sample homogenizer for each tube.

5. Residual protein content

The principle is as follows: under alkaline conditions, the protein converts Cu into²⁺Reduction to Cu⁺，Cu⁺And forming a bluish purple complex with the BCA reagent, measuring the absorbance of the complex at 562nm, and comparing the absorbance with a standard curve to calculate the concentration of the protein to be detected.

And (3) determination of a sample: shearing a sample, weighing 20mg of sample fragments, placing the sample fragments into a homogeneous sample tube (8 built-in 3mM magnetic beads), adding 1mL of lysate containing PMSF with the final concentration of 1mM, and setting the homogenization speed: 6m/sec, time: samples were prepared as suspensions 30sec × 2 times, with temperature control below 37 ℃. The mixture is lysed at 25 +/-5 ℃ for 3h, and the sample is shaken to be mixed evenly every 30 min. Centrifuging at 4000rpm for 10min after cracking, and taking supernatant as test solution. The same method is used for preparing 3 parallel samples.

Measuring 0.4mL of test solution, placing the test solution in 3 clean penicillin bottles, sucking 4mL of BCA working solution by a pipettor, tightly covering the BCA working solution by a rubber plug, uniformly mixing the solution by a vortex oscillator, placing the mixture in a water bath at 37 ℃ for 22min, and immediately measuring the absorbance at 562nm by an ultraviolet spectrophotometer.

And (4) calculating a result:

calculating the protein concentration of the test solution according to the absorbance and the standard curve, and converting the residual protein content (mg/g) in the sample according to the following formula:

wherein C is the content (mg/g) of residual protein in the sample;

C_tsthe final concentration (mg/mL) of residual protein in the test solution;

v is the volume of lysate added to the homogeneous sample tube (1 mL in the formula);

m is the mass (g) of the sample.

6. pH of the leach liquor

Taking a sample, adding normal saline as leaching medium, and when the thickness of the sample is less than or equal to 1.0mm, the surface area of the sample/the volume of leaching liquor is 3cm²/mL，Thickness of sample>1.25cm for 1.0mm sample surface area/volume of leach liquor²mL, extracted at 37 ℃ for 72 hours, to prepare a test solution, and the pH was measured.

The results of the above tests are shown in table 2 below:

TABLE 2

Animal experiments:

28 beagle dogs (male and female are not limited, age 12 months, weight 15.6 + -0.5 kg) with mature bone were used. Groups were divided into four groups according to predetermined post-operative observation times (0, 6, 12 and 24 weeks), and 7 animals were randomly assigned to each group. Randomly selecting one forelimb of each animal to perform rotator cuff bridging operation to serve as an experimental group; the other side was untreated and served as a normal control.

Beagle dogs were placed in lateral decubitus, anesthetized by Shutai vein, and randomly prepared on left or right shoulder joints. The non-skin preparation side is a normal control side and is not subjected to any treatment; the skin preparation side is the experimental side of the operation. The operation process is as follows: a 4-5cm incision was made in the skin above the shoulder and the deltoid muscle was dissected to separate the muscle parallel to the muscle fibers to expose the infraspinatus tendon. The tendon was cut sharply away from the greater tuberosity and transected at the tendon junction, and the infraspinatus tendon was completely excised from the tendon junction of the greater humerus tuberosity, resulting in a defect approximately 20mm in length. The excised portion of the tendon was removed with the capsule, completely exposing the glenoid joint. Two 5X 5mm bone tunnels were made beside the greater tubercle, 5mm apart. One sheet of the rotator cuff patch (length: 50mm, width: 30mm, thickness: 1.6mm) provided in example 1 was cut into the shape shown in fig. 1, wherein A, B are opposite sides of the rotator cuff patch. The rotator cuff patch was fixed to the humeral head at side a through a bone tunnel and sutured to the muscle tissue at side B as shown in fig. 2. The wound was sutured intermittently layer by layer with tenacious 4-0vicryl absorbable sutures and skin was sutured continuously with non-absorbable sutures. 8mg/kg of ceftiofur sodium is injected into beagle dogs intramuscularly after operation for 5 days.

After reaching the predetermined post-operative observation time, the sample was taken (the sampling site is shown in FIG. 3), and biomechanical examination and histological examination were performed. Of the animals at all time points, 5 were subjected to biomechanical testing and 2 were subjected to histological testing.

The general examination results of the animal experiments are as follows:

1. state of health

All animals were able to walk and bear weight immediately after surgery, had no wound infection, no statistical differences in mean body weight, no extravagant death, and good skin healing.

2. Tendon cross-sectional area

The tendon cross-sectional area was calculated by considering the cross-section as a rectangle and measuring the width and thickness of the tendon in the middle region, and the results are shown in table 2 below.

TABLE 2 tendon cross-sectional area (standard deviation) at different time points

As can be seen from table 2, the tendons on the operative side were significantly thicker than the normal side (P <0.01, with very significant statistical significance) at 6 weeks, 12 weeks and 24 weeks after the operation.

3. Tendon rupture site

Biomechanical index was measured on a tensile machine using 25 specimens obtained at 4 time points of 0 week (normal side and operation side), 6 week (operation side), 12 week (operation side) and 24 week (operation side). The fracture sites of concern are mainly muscle sites, muscle tendon junctions, mid tendon and tenosynovium interfaces. The test results are shown in table 3 below:

TABLE 3 statistics of tendon rupture sites

As can be seen from table 3, at 0 weeks post-surgery, 5 specimens on the normal side all broke at the muscle and the operative side all at the aponeurosis interface; after 6 weeks, 3 samples are fractured in the middle section of the tendon, and 2 samples are fractured at the interface of the tendon and the bone; after 12 weeks, 4 samples are fractured in the middle ligament, and 1 sample is fractured in the muscle; at 24 weeks, all samples had fractures similar to the normal control side, with fractures in the muscle, indicating that this index of the regenerated tendon returned to normal levels.

4. Surgical side humeral profile

Fig. 4 is a cross-sectional view of the humerus on the operative side at 6 weeks (a), 12 weeks (B), and 24 weeks (C) after surgery. As can be seen, at each time point, the tendon formed a tenascin interface at the bone tunnel entrance and surrounding cortex (see the star marks in the figure). At

weeks

6 and 12, rotator cuff patch material was visibly evident due to the lower mechanical tension in the bone tunnel (part shown by arrows).

5. Maximum force of breaking

The instantaneous tensile force at which the sample is pulled off on a tensile machine is the maximum force (unit: N) at which the sample is pulled off. The results of the maximum force measurements on the normal and surgical side samples 6-24 weeks after rotator cuff patch implantation are shown in fig. 5. Wherein, 6 weeks after operation, the maximum force of the sample at the operation side is 140.54 + -23.06N, which is 40.15% of that at the normal control side (354.96 + -79.30N); 12 weeks post-surgery, the maximum force of the surgical side sample was 188.04 ± 51.31N, which is 42.76% of that of the normal side (443.89 ± 62.22N); and 24 weeks after surgery, the maximum force of the surgical side sample was 506.72 + -129.36N, with no statistical difference (P >0.05) compared to the normal side (485.54 + -148.66N), which is 95% -100% of the normal side.

6. Tensile strength

Tensile strength is the maximum force per unit area of the sample (in MPa). The results of measuring the tensile strength of the samples on the normal side and the surgical side 6 to 24 weeks after the implantation of the rotator cuff patch are shown in FIG. 6. As can be seen from FIG. 6, since the tendon cross section on the operation side is thicker than that on the normal control side, the corresponding tensile strength is lower than that on the normal control side. Wherein, the tensile strength of the samples on the operation side is 24.25%, 15.28% and 49.98% of the samples on the normal control side respectively at 6 weeks, 12 weeks and 24 weeks after the operation. It can be seen that the tensile strength of the surgical side sample is gradually increasing.

7. Histological examination

Histology was assessed in 2 animals per group at 6, 12 and 24 weeks post-surgery. Harvesting the tendon and regenerated tendon from the proximal end of the tendon junction on the humerus (including the greater tuberosity); contralateral normal control specimens were also collected to check the histological appearance of the tendon under the spinatus. The shoulder joint specimen was dissected longitudinally to fully expose the repaired tissue, muscle and tendon-bone binding interface. Fixation with 10% neutral formalin and decalcification treatment with 5% nitric acid solution were carried out. All histological sections were HE and trichrome stained and viewed under an optical microscope.

1) FIG. 7 is an optical micrograph of HE staining and Masson staining of regenerated tendon tissue in the joint cavity at each observation time after the scheduled surgery. As can be seen in fig. 7, all the surgical side tendinous tissue has formed since week 6 post-surgery. Infiltration of inflammatory cells was seen near the surgical suture on the muscle side. The rotator cuff patch material has been substantially completely degraded.

2) Fig. 8 is an HE stained optical micrograph of regenerated tendon bone healing structures at each observation time after a predetermined period of operation, wherein 6 weeks after operation: a (× 20) and D (× 40); 12 weeks after surgery: b (× 20) and E (× 40); 24 weeks after surgery: c (. times.20) and F (. times.40). As can be seen in fig. 8, at the tenascin interface, a four-layered tenascing structure occurs mainly at the entrance of the bone tunnel and at the cortex surrounding it. Typical tenosynostosis consists of four layers of tissue, organized sequentially tendon tissue and Sharpy's fibers, non-calcified fibrous structures, calcified fibrous structures and bone tissue, respectively. From week 6, it was clearly resolved under microscope. 6-12 weeks after the operation, rotator cuff patch residue and chronic inflammatory mediators can be seen in the marrow tract; and at week 24, the bone tunnels consisted of disorganized collagen-like structures.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the scope of the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The application of the hydrophilic electrospun biological composite scaffold material in preparing the implant for treating rotator cuff tissue tearing is characterized in that the composite scaffold material is prepared by blending a water solution of fibrinogen, L-arginine or hydrochloride thereof and a P (LLA-CL) solution and adopting an electrospinning technology; wherein the mass ratio of the fibrinogen to the L-arginine or the hydrochloride thereof is 1.2: 1-12.5: 1;

the fibrinogen and the L-arginine or the hydrochloride aqueous solution thereof, wherein the solvent is selected from one or more of pure water, water for injection, a salt solution and a buffer solution; the salt solution is selected from sodium chloride solution and potassium chloride solution; the buffer solution is selected from phosphate buffer solution, Tris-HCl buffer solution, glycine buffer solution and D-Hank's solution.

2. Use according to claim 1, wherein the fibrinogen is of mammalian origin.

3. The use according to claim 2, wherein the mammal is a human, pig, cow, sheep or horse.

4. The use according to claim 1, wherein the mass ratio of polylactic acid to polycaprolactone in P (LLA-CL) is 20:80 to 95: 5.

5. The use according to claim 1, wherein the solvent in the solution of P (LLA-CL) is selected from one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran, chloroform or acetone.

6. The use according to claim 1, wherein the mass ratio of fibrinogen to P (LLA-CL) is 0.2:1 to 2.1:1 after blending the aqueous solution of fibrinogen, L-arginine or hydrochloride thereof with the solution of P (LLA-CL).

7. The use according to claim 1, wherein the hydrophilic electrospun biocomposite scaffold material has an equilibrium contact angle of less than 55 °.

8. The use of claim 1, wherein the hydrophilic electrospun biocomposite scaffold material has a total volume shrinkage of no more than 20% after contact with an aqueous solution; the porosity is not less than 30%.

9. Use according to claim 1, wherein the aqueous solution of fibrinogen, L-arginine or hydrochloride thereof is further loaded with an antibacterial substance selected from one or more of the group consisting of penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, macrolides, glycosides, sulfonamides, quinolones, nitroimidazoles, lincosamines, fosfomycin, chloramphenicol, para-colistin B, bacitracin.

10. Use according to claim 9, wherein the penicillins are selected from the group consisting of penicillin, ampicillin, carbenicillin; the cephalosporins are selected from cefalexin, cefuroxime sodium, ceftriaxone and cefpirome; the carbapenem is thiomycin; the aminoglycoside is selected from gentamicin, streptomycin, and kanamycin; the tetracycline is selected from tetracycline and chlortetracycline; the macrolides are selected from erythromycin and azithromycin; the glucoside is vancomycin; the sulfonamides are selected from sulfadiazine and trimethoprim; the quinolone is selected from the group consisting of pipemidic acid and ciprofloxacin; the nitroimidazoles are selected from metronidazole and tinidazole; the lincomycin is selected from lincomycin and clindamycin.

11. Use according to claim 9 or 10, wherein the amount of said antibacterial substance released is not less than 30% of the total loading within 15 minutes after implantation of the scaffold material in the body.

12. A rotator cuff patch, wherein the rotator cuff patch has a three-dimensional nano-network structure and is made of a raw material comprising a fibrinogen complex and polylactic acid-caprolactone.

13. The rotator cuff patch according to claim 12, wherein the fibrinogen complex comprises the following components in parts by weight: 0.1-20 parts of fibrinogen, 0.1-10 parts of arginine hydrochloride, 0.01-10 parts of sodium chloride and 1-10 parts of sodium citrate;

preferably, the fibrinogen complex comprises the following components in parts by weight: 3-15 parts of fibrinogen, 0.5-5 parts of arginine hydrochloride, 0.3-5 parts of sodium chloride and 1-10 parts of sodium citrate.

14. The rotator cuff patch according to claim 12 or 13, wherein the polylactic acid-caprolactone has Mn of 30000 and 80000, Mw of 80000 and 160000, and Mw/Mn of < 3.0;

preferably, the mass ratio of the polylactic acid chain segment to the polycaprolactone chain segment in the polylactic acid-caprolactone is 20: 80-95: 5.

15. The rotator-cuff patch according to any one of claims 12 to 14, wherein the rotator-cuff patch has a thickness of 1.6 to 10mm, preferably 2 to 3 mm.

16. The rotator cuff patch according to any one of claims 12 to 15, wherein the rotator cuff patch has a protein content of 100-220mg/g and a residual protein content of <12 mg/g;

preferably, the pH of the water extract of the rotator cuff patch is 6-8.

17. The rotator cuff patch according to any one of claims 12 to 16, wherein the rotator cuff patch has a porosity of 41 to 100%, preferably 41 to 80%;

and/or the water absorption rate of the rotator cuff patch is 35-200%, preferably 55-80%;

and/or the tensile strength of the rotator cuff patch is 2-5 MPa;

and/or the rotator cuff patch has an elongation at break of 100-200%.

18. The rotator cuff patch according to any one of claims 12 to 17, further comprising an antibacterial and/or anti-inflammatory drug.

19. A method of manufacturing a rotator cuff patch according to any one of claims 12 to 18, wherein the method comprises the steps of:

20. The method according to claim 19, wherein the concentration of the polylactic acid-caprolactone solution is 5-8 g/100 mL;

preferably, the solvent of the polylactic acid-caprolactone solution is selected from one or a combination of at least two of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran and acetone;

preferably, the solvent of the fibrinogen complex solution is water or an aqueous solution;

preferably, the concentration of the fibrinogen complex solution is 8-29g/100 mL;

preferably, the mass ratio of the fibrinogen compound to the polylactic acid-caprolactone in the fibrinogen compound solution and the polylactic acid-caprolactone solution is 0.48-1.1: 1.

21. The method of claim 19, wherein the electrospinning conditions are: the voltage difference is 15-140kV, the flow rate of the solution is 3-399mL/h or 401-960mL/h, and the distance between the needle head and the mandrel is 10-50 cm;

preferably, the preparation method further comprises: and the surface of the rotator cuff patch is coated with a medicament with antibacterial and/or anti-inflammatory effects.