CN114452442A

CN114452442A - Implant for in-situ induction of uterine wall tissue regeneration

Info

Publication number: CN114452442A
Application number: CN202210114599.6A
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: 2022-05-10
Also published as: WO2023143336A1

Abstract

The invention discloses an implant for in-situ induction of uterine wall tissue regeneration, which is prepared from raw materials comprising a fibrinogen compound and a polylactic acid polycaprolactone copolymer, and a three-dimensional nano-net structure is arranged in the implant. By implanting the implant of the invention into the uterine wall incision under the serosa, the regeneration of uterine wall tissues (including but not limited to serosa, uterine smooth muscle, endometrium, connective tissue and extracellular matrix) after cesarean section can be effectively induced in situ, the formation of scar tissues is prevented, and further, a plurality of long-term adverse reactions caused by cesarean section defects are fundamentally prevented.

Description

Implant for in-situ induction of uterine wall tissue regeneration

Technical Field

The invention relates to the technical field of medicine, in particular to an implant for in-situ inducing uterine wall tissue regeneration.

Background

Cesarean section (cesarean section CS) is an effective means to deal with high-risk pregnancies and to solve dystocia, saving the lives of pregnant and lying-in women and perinatal infants. After 3 centuries of exploration, the cesarean section is gradually developed into a mature operation, but with the development of society and economy, the significance of the cesarean section operation is gradually distorted by people. Global population-based caesarean section yields are increasing year by year, with hospital-based caesarean sections increasing in most countries of the world, including sub-saharan africa countries. The study of Dublin in Ireland showed that the caesarean section yield rose from 18.3% to 23.5% in 2005-2014. The results of the barlestan study showed a caesarean section yield of 22.9%. The caesarean yield in egypt is as high as 55%. The average caesarean section yield in China in 2011 reaches 54.472%, and even reaches 71.588% in some regions.

The literature reports that the incidence of cesarean section defect (cesarean section defect CSD) after one cesarean section is 61%, the incidence rate rises to 81% after two CSs, and even reaches 100% after three CSs. In a group of women receiving urgent CS treatment, women with CSD have a longer duration of labor. Peripheral infections, increased body mass index and diabetes are also associated with CSD. It was found that the later the CS delivery, the greater the risk of developing greater CSD, and if delivery was continued for 5 hours or the cervix dilated 5cm, the chance of developing greater CSD increased significantly. The incidence of retroflexed uterus is even higher than that of anteflexed uterus. Recently, it has also been found that a large incidence of CSD is 6 times higher in lower hysterectomies (less than 2cm) compared to higher hysterectomies (2 cm above the bladder uterine sac).

The cesarean section can cause great influence on the near and far period of maternal operation. The recent effects are mainly: 1) hypotension syndrome in supine position, 2) hemorrhage, 3) organ injury in adjacent organs, 4) amniotic fluid embolism, etc.; the long-term effects are mainly: 1) pelvic cavity adhesion; 2) endometriosis, 3) uterine rupture during second pregnancy, 4) implantation of a pre-placenta and placenta during second pregnancy; 5) pregnancy is carried out at scar position of uterine incision after cesarean section.

Most CSD is asymptomatic, but may still be associated with complications of late pregnancy such as uterine rupture, abnormal placental adhesion or scar rupture. Significant pathological changes include lower uterine segment distortion and widening, endometrial congestion, polyps, lymphocyte infiltration, residual sutures, telangiectasia, free red blood cells, scar endometrial fragmentation and rupture, and iatrogenic adenomyosis. Most excised specimens show only fibrotic tissue. The incidence of endometriosis at the deepest point of CSD was 27.2% and 21.1%, respectively. This not only causes pain and dysmenorrhea, but also blood retention due to abnormal bleeding. Ectopic pregnancy occurred in 6% of pregnant women with a history of cesarean section. Including pregnancy where the myoma of the uterus is scarred prior to implantation. The pregnancy is located on the scar, surrounded by the muscular layer and connective tissue. The mechanism of implantation at this site is thought to be migration of the embryo through the lower uterine wedge defect or through a microscopic fistula. In symptomatic patients, clinical manifestations range from painful or painless vaginal bleeding to uterine rupture and hypovolemic shock. Diagnosis is by ultrasound (transvaginal and transabdominal) and is seen with enlarged hysterectomy scar, with buried masses that extend beyond the anterior contour of the uterus. There is no consensus on the best treatment of Cesarean Scar Pregnancy (CSP). Treatment options include laparotomy or laparoscopic wedge resection of ectopic pregnancy, or possibly hysterectomy, dilation, curettage, or methotrexate treatment. In subsequent pregnancies, recurrent scar implantation may occur.

It follows that the main cause of CSD is due to scar healing in caesarean section uterine incisions. How to prevent the scar formation is a key problem for fundamentally preventing the CSD.

Disclosure of Invention

The invention aims to provide a hydrophilic electrostatic spinning implant for preventing and treating caesarean section defects, a preparation method thereof and application of the implant in preventing scarring of uterine walls.

In a first aspect, the present invention provides an implant for inducing uterine wall tissue regeneration in situ, the implant having a three-dimensional nano-network structure therein and being made of a raw material comprising a fibrinogen complex and a polylactic acid-polycaprolactone copolymer.

In some embodiments, the fibrinogen complex includes 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.

For example, the fibrinogen may be 0.1, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15, 18, or 20 parts, etc.

For example, arginine hydrochloride can be 0.1, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts, and the like.

For example, the sodium chloride can be 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts, and the like.

For example, the sodium citrate may be 1 part, 1.2 parts, 1.5 parts, 1.8 parts, 2 parts, 2.2 parts, 2.5 parts, 2.8 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or the like.

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.

Preferably, the fibrinogen is mammalian-derived fibrinogen; the mammal comprises human, pig, cattle, sheep or horse; further preferably, the fibrinogen is porcine blood-derived fibrinogen.

In the raw material of the implant, the mass ratio of the fibrinogen compound to the polylactic acid-polycaprolactone copolymer is (0.48-1.1): 1. Specifically, the mass ratio of the two may be 0.48:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.84:1, 0.9:1, 1:1, 1.08:1, 1.1: 1.

In some embodiments, the polylactic acid polycaprolactone copolymer has a molecular weight of 5 to 30 tens of thousands, for example, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, 130000, 140000, 150000, 160000, 170000, 180000, 190000, 200000, 210000, 220000, 230000, 240000, 250000, 260000, 270000, 280000, 290000, or 300000, and the like.

In some embodiments, the protein content of the implant is 100-220 mg/g, for example, can be 100mg/g, 120mg/g, 130mg/g, 150mg/g, 160mg/g, 180mg/g, 200mg/g, or 220mg/g, etc.; 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, or 1 mg/g. Wherein, the protein content is the protein content measured in a test solution prepared by dissolving a sample in a sodium hydroxide solution; the residual protein content is the protein content measured in a test solution obtained by leaching a sample with water for injection or physiological saline.

In some embodiments, the implant is in the form of a membrane.

In some embodiments, the thickness of the implant is 0.51mm to 1.4mm, e.g., 0.51mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, etc., preferably 0.6mm to 0.8 mm.

In some embodiments, the porosity of the implant is 41% to 80%, such as 41%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, etc., preferably 45-70%.

In some embodiments, the water absorption of the implant is 35% to 200%, e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 140%, 160%, 180%, 200%, etc., preferably 55% to 80%.

In some embodiments, the implant has a tensile strength of 0.5 to 5.0MPa, such as 0.5MPa, 1.0MPa, 1.5MPa, 2.0MPa, 2.5MPa, 3.0MPa, 3.5MPa, 4.0MPa, 4.5MPa, 5.0MPa, and the like, preferably 1.0 to 4.0 MPa.

In some embodiments, the implant has an elongation at break of 60% to 200%, such as 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, etc., preferably 70% to 160%.

In some embodiments, the implant is made using electrospinning.

In some embodiments, when the implant is prepared, the solution containing the fibrinogen complex and the solution containing the polylactic acid-polycaprolactone copolymer are uniformly mixed and added into the same volumetric tube of an electrostatic spinning machine for electrostatic spinning preparation; or adding the solution containing the fibrinogen compound and the solution containing the polylactic acid-polycaprolactone copolymer into two different volumetric tubes of an electrostatic spinning machine respectively, and carrying out electrostatic spinning simultaneously.

In some embodiments, the solution containing the polylactic acid-polycaprolactone copolymer is prepared by dissolving the polylactic acid-polycaprolactone copolymer in a mixed solvent of one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran and acetone at a mass volume percentage concentration of 5% to 8% (i.e., 5 to 8g/100 mL). For example, the concentration of the polylactic acid polycaprolactone copolymer may be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%.

In some embodiments, the fibrinogen complex-containing solution is prepared by dissolving the fibrinogen complex in distilled water at a mass volume percent concentration of 8.0% to 29.0% (i.e., 8.0 to 29.0g/100mL), for example, the mass concentration may be 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%.

In some embodiments, the residual amount of solvent in the implant is <0.55 mg/g.

In some embodiments, the voltage difference of the electrostatic spinning is 15-140 Kv, and/or the electrospinning distance is 10-50 cm, and/or the electrospinning liquid advancing speed is 3-399mL/h and 401-960 mL/h.

For example, the voltage difference of the electrospinning may be 15kV, 20kV, 30kV, 40kV, 50kV, 60kV, 70kV, 80kV, 90kV, 100kV, 110kV, 120kV, 130kV, 140kV, or the like.

For example, the electrospinning distance may be 10cm, 20cm, 30cm, 40cm, or 50cm, etc.

For example, the electrospinning liquid advancing speed may be 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, 960mL/h, and the like.

In a second aspect, the present invention provides a method of implanting the implant, comprising the steps of: the implant is placed between the muscularis lining of the uterine wall or between the serosal layer and the muscularis lining of the uterine wall.

In a third aspect, the present invention provides the use of the implant in the manufacture of a material for repairing a defect in a body tissue.

In some embodiments, the material is a repair material for treating meninges, abdominal defects, pelvic floor organ prolapse, atria, ventricular septum, pericardial defects, tendon or ligament rupture, or parenchymal organ rupture.

In a fourth aspect, the invention provides the use of said implant in the manufacture of a material for use in the in situ induction of regeneration of uterine wall tissue or the prevention of scar tissue formation or the prevention and treatment of post-operative complications of the uterus.

In some embodiments, the uterine wall tissue includes, but is not limited to: serosa, uterine smooth muscle, endometrium, connective tissue, extracellular matrix, and the like.

In some embodiments, the complications include pelvic adhesions, endometriosis, uterine rupture upon re-pregnancy, pre-placenta upon re-pregnancy, placenta implantation, distant adverse reactions such as pregnancy at the scar site of a uterine incision from caesarean section, and the like.

In a fifth aspect, the present invention provides a use of a hydrophilic electrospun biological composite scaffold material in the preparation of a material for inducing the regeneration of uterine wall tissue in situ or preventing scar tissue formation or preventing and treating complications after uterine surgery, the composite scaffold material is prepared by blending an aqueous solution of fibrinogen, L-arginine or hydrochloride thereof with a P (LLA-CL) solution, and preparing the same by 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 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, the uterine wall tissue comprises serosa, uterine smooth muscle, endometrium, connective tissue, and extracellular matrix.

In some embodiments, the complications include pelvic adhesions, endometriosis, uterine rupture upon re-pregnancy, pre-placenta upon re-pregnancy, placenta implantation, distant adverse reactions such as pregnancy at the scar site of a uterine incision from caesarean section.

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

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

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

In some embodiments, 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, the fibrinogen, L-arginine, or hydrochloride salt thereof in an aqueous solution is blended with a 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, the hydrophilic electrospun biocomposite scaffold material has an equilibrium contact angle of less than 55 °.

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

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

In some embodiments, 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 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.

In some embodiments, 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 has at least one of the following beneficial effects:

the implant provided by the invention is a hydrophilic electrospun biological composite stent implant, can prevent the cicatrization of the uterus, can be used as a uterus patch, and can prevent and treat the cicatrization uterus by implanting the implant into a wound surface formed by a caesarean section incision. The implant is prepared by adopting a soft tissue inductive biomaterial, is prepared by taking a denatured natural polymer material and a degradable synthetic polymer material as raw materials through an electrostatic spinning technology, and has a three-dimensional nano-mesh structure. The implant does not contain bioactive factors and living cells, and does not need any chemical or biological crosslinking agent for crosslinking and fixing.

The invention uses the blend of fibrinogen and degradable high molecular material L-lactide, caprolactone copolymer (Fg/PLCL), and adopts the electrostatic spinning technology to prepare the super hydrophilic biodegradable composite reticular stent material. We surprisingly found that by implanting the material into a uterine wall incision under serosa, the regeneration of the uterine wall incision tissue (comprising smooth muscle, fibroblasts and vascular endothelial cells) of the labrador retriever can be effectively induced in situ, so that the recovery of the function and the structure of the uterine wall is realized, and the occurrence of long-term adverse reactions after cesarean section is prevented. In addition, the electrostatic spinning technology is adopted to manufacture the implant material, 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, degradation rate, thickness, porosity, water absorption 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 shows a surgical bioprosthetic patch (dedicated to uterine wall regeneration).

Fig. 2 shows the uterine wall HE staining of normal dogs.

Figure 3 shows control groups post-operatively 4 weeks (a, C) and 8 weeks (B, D) HE staining. Wherein: a and B are respectively 40 ×; c and D are 100X. Arrows indicate sutures.

Figure 4 shows post-operative 4 weeks (a, C) and 8 weeks (B, D) HE staining in the experimental groups. Wherein: a and B are 40X respectively; c and D are 100X. The triangles indicate patch patches; arrows indicate sutures.

Figure 5 shows a comparison of endometrial thickness at 4 and 8 weeks post-surgery for the experimental and control groups.

FIG. 6 shows Masson staining of the uterine wall of a normal dog.

Figure 7 shows Masson staining at 4 weeks (a, C) and 8 weeks (B, D) post-surgery in the control group. Wherein: a and B are 40X respectively; c and D are 100X. Arrows indicate sutures.

Fig. 8 shows Masson staining at 4 weeks (a, C) and 8 weeks (B, D) post-operatively in the experimental groups. Wherein: a and B are 40X respectively; c and D are 100X. The triangles indicate patch patches; arrows indicate sutures.

Fig. 9 shows the area ratio of smooth muscle regions in the uterine operated region at 4 weeks and 8 weeks after the operation.

FIG. 10 shows microvascular density at different remodeling times for a full thickness lesion of the uterine wall.

FIG. 11 shows the density of proliferating cells (400X cells/field) at different healing times for a full-thickness lesion in the uterine wall.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

In this example, a polylactic acid polycaprolactone/fibrinogen (PLCL/Fg) surgical bioprosthetic patch (for uterine wall regeneration) was prepared by electrospinning.

Polylactic acid polycaprolactone copolymer (molecular weight 20 ten thousand) was dissolved in Hexafluoroisopropanol (HFIP) at a concentration of 6%, and fibrinogen complex (7.83 parts of fibrinogen, 1.8 parts of arginine hydrochloride, 2.2 parts of sodium chloride, and 6.17 parts of sodium citrate) was dissolved in distilled water at a concentration of 18%. Mixing and stirring a solution of polylactic acid-polycaprolactone copolymer and a solution of a fibrinogen compound (the mass ratio of the fibrinogen compound to the polylactic acid-polycaprolactone copolymer is 0.9: 1), preparing a hydrophilic electrostatic spinning implant by an electrostatic spinning machine, wherein the voltage difference of electrostatic spinning is 100Kv, the electrospinning distance is 20cm, the advancing speed of electrostatic spinning solution is 200mL/h, sterilizing by adopting 25KGy electron beams, packaging by vacuum nitrogen charging, and storing at 2-8 ℃.

By adjusting electrostatic spinning parameters, the embodiment specifically provides 6 hydrophilic electrostatic spinning implants with different thicknesses, the obtained implants are in a membrane shape, the thicknesses are respectively 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm and 1.0mm, the corresponding porosity is in a range of 45% -70%, the water absorption is in a range of 55% -80%, and the tensile strength is in a range of 2.0-4.0 MPa.

Example 2

Polylactic acid polycaprolactone copolymer (molecular weight 30 ten thousand) was dissolved in chloroform at a concentration of 5%, and fibrinogen complex (fibrinogen 6.09 parts, arginine hydrochloride 2.0 parts, sodium chloride 2.0 parts, and sodium citrate 3.91 parts) was dissolved in distilled water at a concentration of 14%. Mixing and stirring a solution of a polylactic acid-polycaprolactone copolymer and a solution of a fibrinogen composite (the mass ratio of the fibrinogen composite to the polylactic acid-polycaprolactone copolymer is 0.84: 1), preparing a hydrophilic electrospun implant by using an electrospinning machine, sterilizing by adopting a 25KGy electron beam at an electrospinning voltage difference of 50Kv and an electrospinning distance of 10cm at an electrospinning liquid advancing speed of 100mL/h, vacuum nitrogen-filled packaging, and storing at 2-8 ℃.

By adjusting the electrospinning parameters, the embodiment specifically provides 5 hydrophilic electrospun implants with different porosities, the obtained implants are in a membrane shape, the porosities are 41%, 45%, 50%, 70% and 80%, the corresponding thicknesses are in the range of 0.6mm to 0.8mm, the water absorption is in the range of 50% to 100%, and the tensile strength is in the range of 2.0 to 4.0 MPa.

Example 3

Polylactic acid polycaprolactone copolymer (molecular weight 5 ten thousand) was dissolved in tetrahydrofuran at a concentration of 8%, and fibrinogen complex (fibrinogen 12.61 parts, arginine hydrochloride 3.29 parts, sodium chloride 3.27 parts, and sodium citrate 9.83 parts) was dissolved in distilled water at a concentration of 29%. Mixing and stirring a solution of polylactic acid-polycaprolactone copolymer and a solution of a fibrinogen compound (the mass ratio of the fibrinogen compound to the polylactic acid-polycaprolactone copolymer is 1.08: 1), preparing a hydrophilic electrostatic spinning implant by an electrostatic spinning machine, wherein the voltage difference of electrostatic spinning is 100Kv, the electrospinning distance is 30cm, the advancing speed of electrostatic spinning liquid is 500mL/h, sterilizing by adopting 25KGy electron beams, vacuum nitrogen-filled packaging, and storing at 2-8 ℃.

By adjusting the electrospinning parameters, the embodiment specifically provides 6 hydrophilic electrospun implants with different water absorption rates, the obtained implant is in a membrane shape, the water absorption rates are 55%, 65%, 75%, 80%, 90% and 100%, the corresponding thicknesses are in the range of 0.6mm to 0.8mm, the porosity is in the range of 45% to 70%, and the tensile strength is in the range of 3.0 to 4.0 MPa.

Experimental example 1

1. Materials and methods

1.1 materials and reagents

Polylactic acid polycaprolactone copolymers (PLCL) are derived from Purace Biomaterials (Gorinchem, the Netherlands). Porcine fibrin (fibre gen, Fg) was prepared according to the formulation and process disclosed in CN 101759766A. All other reagents and chemicals, except where specifically noted, were purchased from Sigma-Aldrich (st louis, usa).

1.2 preparation of surgical BioPatch (for uterine wall regeneration) samples

The polylactic acid polycaprolactone/fibrinogen (PLCL/Fg) surgical biological patch (used for uterine wall regeneration, hereinafter referred to as uterine patch) is prepared by an electrostatic spinning method. The fibrinogen complex was dissolved in distilled water at a concentration of 18% and the PLCL was dissolved in Hexafluoroisopropanol (HFIP) at a concentration of 8% according to the procedure of example 1. The two solutions were mixed and stirred at a ratio of 0.23:0.77 and the sample for investigation was prepared by electrostatic spinning (model: NS1WS 500Elmarco Czech). Sterilizing by adopting 25KGy electron beams, vacuum nitrogen-filled packaging, and storing at 2-8 ℃.

1.3 design of the experiment

The study was approved by my animal care and use committee. 6 labrador retrievers (female, older than 12 months; weighing 15.6 + -0.5 kg) with mature skeletal development were used. Three groups were divided according to the predetermined post-operative observation time (4 weeks, 8 weeks), and 3 animals were randomly assigned to each group. Self-pairing is adopted, three different treatments are respectively carried out on the uterus of each animal, and then the pathological effect analysis after implantation is carried out. The grouping of the uteri in each group is shown in table 1.

TABLE 1 grouping of uterine regeneration

Follow-up time (week)	Artificial operation group	Control group	Test group
					4	3	3	3
8	3	3	3
				Total up to	6	6	6

Fasting was performed before surgery. Sutai 50 (Vickers, France, batch No. 7VU4A)25mg/kg intramuscular injection, and xylazine hydrochloride (Gilin Hua Murray, Seiko No. 20201118)5mg/kg intramuscular injection for anesthesia, optionally supplemented with an appropriate amount of isoflurane. After anesthesia, the animals were placed in the supine position, the limbs were properly fixed on the operating table, the lower abdomen was prepped, and a conventional sterile drape was used. And (3) taking down the median longitudinal incision of the abdomen, cutting off the skin and all layers of the abdominal wall in sequence to enter the abdominal cavity, wherein the distance is about 2cm from the pubic symphysis and the length is about 4-5 cm. The uterus is exposed between the bladder and rectum. Two parallel incisions perpendicular to the longitudinal axis of the uterus are made in the middle section of the uterus, which is about 1cm long and reaches the inside of the uterine cavity. The distance between the two cuts is more than 1.5 cm. One uterine wall incision was randomly selected for each animal as a test group, and double-layer suturing was performed using 6-0prolene thread, in which a 1X 1.5cm uterine patch was placed under the serosa incision. The other parallel incision was a control group. The test group was identical except that no patch was placed under the serosa. After operation, the injection of penicillin is continuously carried out for 5 days, 80 million U/day of penicillin is injected, and the infection is prevented by 10 million U/day of gentamicin. Observing the mental state, food and drink condition and excrement and urine condition of the animals after the operation. At predetermined time points as sampling, anatomical and histological examination.

After 4 weeks and 8 weeks of postoperative feeding, making lower abdominal incision under anesthesia, exposing uterus, finding out operation area, observing whether adhesion exists between operation area and surrounding tissue, and whether stenosis and hydrops exist in uterus, and shooting with digital camera.

1.4 a-SMA immunohistochemical staining

The content of the uterine smooth muscle of the dog is detected by using rabbit anti-a-SMA polyclonal antibody. The method comprises the following steps: 1) dewaxing and hydrating: and baking the tissue slices in a 95 ℃ drying instrument for 30 min. Xylene (I)5 min-xylene (II)5 min-xylene (III)5 min-absolute ethanol (I)3 min-95% ethanol 3 min-85% ethanol 3 min-80% ethanol 3 min-running water washing for 30 min. 2) Washing with PBS for 5min for 2-3 times. 3) Antigen retrieval: 2000mL of EDTA antigen retrieval solution (working solution) was injected into a stainless steel pressure cooker, the slices were placed on a metal stand and placed into the cooker (the slices were below the liquid level), and the pressure valve was closed. Heating was started. And (5) after the pressure cooker starts to slowly blow air, reducing the power and timing for 2 min. Cooling the pressure cooker end away from the heat source at room temperature for 30min, taking off the air valve, opening the cooker cover, and cooling the slices to room temperature. The sections were washed once with distilled water and then 3 times with PBS for 2min each. 4) Dropwise adding 3% H₂O₂Incubate at room temperature for 10min to eliminate endogenous peroxidase activity.

1.5 immunohistochemical staining for CD31

The content of uterine microvessels is detected by using a rabbit anti-CD 31 polyclonal antibody. The specific method is the same as the a-SMA immunohistochemical staining process, wherein the primary antibody is diluted by rabbit anti-CD 31 polyclonal antibody 1: 10000. Positive control: positive control human tongue tissue specimens of known positive are used, localized to the cytoplasm; negative control: replacing primary antibody with normal dog serum, and obtaining a negative result; blank control: the primary antibody was replaced with PBS, and the result was negative.

1.6 Ki67 immunohistochemical staining

The primary antibody was diluted 1:1000 with rabbit anti-Ki 67 polyclonal antibody. Positive control: positive control with known positive lymph node tissue, localized to the nucleus; negative control: replacing primary antibody with normal dog serum, and obtaining a negative result; blank control: the primary antibody was replaced with PBS, and the result was negative.

1.7 histological observations

The tissue sections were observed using K-Viewer software (KFBIO Jiangfeng biology, Ningbo, China) and the operative and normal areas were determined at 40 Xs. Within the respective operative and normal areas, 10 fields were randomly taken at 400 × field, and the following cell counts and respective calculations and analyses were performed.

1.7.1 determination of area ratio of surgical region uterine smooth muscle region at different healing time

The selected whole layer area of the uterus was measured randomly. Determining the area of a brown-yellow (a-SMA immunohistochemistry positive) region within each region; and calculating the proportion of the a-SMA immunohistochemical positive staining area of the operation area to the corresponding area of the whole uterus (the operation area and the normal area). Taking the average value as the area ratio of the uterine smooth muscle area in the operation area of the animal.

1.7.2 Density of microvessels in the surgical field

Any brown-yellow endothelial cells or clusters of endothelial cells that do not connect to adjacent vessels, connective tissue within the selected area are counted as an independent microvessel. The mean value was taken as the uterine microvascular density of the animal.

1.7.3 surgical field proliferative cell count

The number of yellow brown nuclei stained positive for Ki67 in the selected area was counted as one proliferating cell. The average value was taken as the density of the cells proliferating in this section.

2. Results of the experiment

2.1 uterine Patch surface morphology

The uterus patch was white in appearance, soft, loose (fig. 1A). Under a scanning electron microscope, the uterus patch is of a nano-scale three-dimensional reticular structure, and the fiber diameter is 100-900 nm. The porosity is 60% -80% (figure 1B).

2.2 Hi staining histological observation of dogs after repair of Whole layer injury to uterine wall

The uterus was opened 4 weeks and 8 weeks after the full-thickness uterine injury in the test dogs, respectively, and the uterus was exposed to observe the healing of the uterus in general. 4 weeks and 8 weeks after operation, abundant capillary vessels can be seen on the tissue surface of the test repair area, and the test repair area can be well fused with surrounding tissues; the tissue surface of the control group repair area is seriously adhered with the surrounding tissues; no infection and no water accumulation.

The uterus histology is divided into three layers, the intima, the muscularis and the adventitia. The endometrium is composed of epithelium and stroma, and the epithelium is a tightly arranged single-layer columnar epithelium capable of forming low fold or fossa glandulae; the interstitium is composed of fibrous connective tissue, with a large number of single-tubular uterine glands, with fewer branches and bends. The myometrium is composed of inner and outer longitudinal smooth muscles, and a loose connective tissue containing many great vessels is between the two muscle layers. The endometrium is the serosal layer (fig. 2).

4 weeks after surgery: the control group had a large amount of collagen deposition in the operative area of the uterine wall, lack of glandular and vascular components, and had discontinuous smooth muscle tissue (fig. 3A, C). The test group showed that the pieces of the patch under the serosa were degrading, the thicker endometrium contained a lot of blood vessels, glands and cells, and the continuous smooth muscle fibers were found under the endometrium, similar to the normal sham operation group. The myometrium is composed of the inner and outer longitudinal smooth muscles (fig. 4A, C). After 8 weeks of surgery, the control group had endometrial scarring, lacked cellular and vascular components, thin layers of non-continuous muscularis, and structural disturbances (fig. 3B, D). The muscle fiber content of the test group is obviously increased, the muscle layer is thicker and continuous, and the inner ring and outer longitudinal two-layer structure similar to the normal uterus is further thickened to be close to the normal sham operation group (figures 4B and D).

Endometrial thickness measurements showed (figure 5): the intima thickness (2459.32 + -268.12 μm) was significantly thicker in the test group than in the control group (1180.21 + -128.42 μm) and was close to that in the sham-operated group (3312.94 + -325.97 μm) at 4 weeks after the operation. The difference in endometrial thickness in the test group compared to the control group was statistically significant. At 8 weeks after surgery, the endometrial thickness increased in each group compared to 4 weeks after surgery, and the experimental group (3295.78 + -181.64 μm) was close to the sham (3627.90 + -277.29 μm) and significantly thicker than the control group (1576.13 + -167.39 μm).

2.3 Masson staining histological observation of dogs after repair of Whole layer injury to uterine wall

The extracellular matrixes such as collagen fibers and the like are observed to be blue by Masson staining; the cytoplasm, muscle fibers and red blood cells are red. The endometrium of the sham operation group is thick, has a large number of cells and abundant blood vessels, and the muscle layer is thick and consists of an inner ring and an outer longitudinal two-layer smooth muscle (figure 6).

The control group had 4 weeks after surgery, had less collagen deposition in the surgical area, less blood vessels and cells, and a discontinuous thin muscular layer was visible (fig. 7A, C). After 8 weeks of surgery, the surgical area was scarred, devoid of blood vessels and cells, with a thin discontinuous muscle layer (fig. 7B, D).

4 weeks after the operation of the test group, the degradation of the serosal patch fragments can be seen, the thicker endometrium of the operation area contains a large amount of blood vessels, glands and cells, and continuous smooth muscle fibers can be seen under the endometrium, which is similar to the normal sham operation group. The myometrium is composed of the inner and outer longitudinal smooth muscles (fig. 8A, C).

After 8 weeks of operation, the content of muscle fiber is obviously increased, the muscle layer is thicker and continuous, and the inner ring and outer longitudinal two-layer structure similar to that of a normal uterus is further thickened to be close to that of a normal false operation group (figures 8B and D).

2.4 uterine wall a-SMA immunohistochemical staining

Smooth muscle stains brown and cell nuclei blue. The normal test dog has a thicker myometrium, which is composed of an inner ring and an outer longitudinal smooth muscle, the inner ring muscle is more developed, the outer longitudinal muscle is thinner, and a loose connective tissue layer is arranged between the two layers of muscles and contains a plurality of large blood vessels. After 4 weeks of operation, immunohistochemical staining of a-SMA revealed that there were few but discontinuous muscle fibers in the surgical field in the control group. The experimental group has more muscle fibers in the operation area, has an inner ring and outer longitudinal two-layer structure, and has obviously higher muscle fiber content than the control group. After 8 weeks. The control group mainly replaced the original normal tissue structure with scar formation, the muscular layer was thin and disordered, and a small amount of the muscular layer remained thin and discontinuous. The muscle layer of the test group is thick and continuous, and an inner ring and an outer longitudinal layer similar to a normal uterus are locally formed.

The measurement results of the area ratio of the smooth muscle region in the operation region showed that (fig. 9): after 4 weeks of operation, the area ratio of the smooth muscle area of the test group is 21.34 +/-8.20 percent, which is obviously higher than that of the control group by 11.11 +/-4.17 percent, and the comparison difference has statistical significance. After 8 weeks, the smooth muscle area of each group increased more than before, and the increase of the test group was more obvious. The area ratio of the smooth muscle area of the test group is 30.78 +/-4.15 percent, which is obviously higher than that of the control group by 18.11 +/-7.25 percent. But significantly below the sham operated area 49.85 + -12.23%.

2.5 microvascular density at different repair times of full-thickness injury of uterine wall

After 4 weeks and 8 weeks, the uterus of each group was subjected to CD31 immunohistochemical staining, and the regeneration of the microvessels of each group was observed. Normal endometrium contains a large number of blood vessels. A large number of brown yellow endothelial cells can be seen by CD31 immunohistochemical staining and are uniformly distributed. At 4 weeks post-surgery, only a small number of scattered capillaries were visible in the control endometrium. The content of blood vessels in the test group is higher than that in the control group, a large amount of capillary angiogenesis can be seen in the endometrium, and the distribution is more uniform and is similar to that of the normal endometrium. The number of capillaries in the control group increased 8 weeks after the operation as compared with the previous one. The number of blood vessels in the experimental group is more than that in the control group, but the distribution is not uniform. The number of endometrium capillary vessels is obviously increased compared with the previous one, and the endometrium capillary vessels are uniformly distributed in the whole endometrium and are similar to the normal endometrium blood vessels.

Statistical results of microvascular density in the surgical field showed (fig. 10): after 4 weeks, the density of the microvessels in the test group (12.86 +/-3.70) is obviously higher than that in the control group (8.68 +/-2.70), and is close to that in the sham operation group. The comparison difference is statistically significant. After 8 weeks, the density of the microvessels in each group is increased compared with that in the previous group, and the increase is more obvious in the test group and is close to that in the sham operation group (17.42 +/-3.6). The number of the capillaries in the test group (16.98 +/-2.10) and the number of the capillaries in the control group (10.94 +/-1.07) have obvious statistical significance.

2.6 number of proliferating cells in operation area after repair of full-thickness injury of uterine wall

After 4 weeks and 8 weeks, the uteri of each group were subjected to Ki67 immunohistochemical staining, and the number of proliferating cells in each group was observed. Normal endometrium cells change along with the cycle of estrus and generate periodic proliferation and apoptosis. Normal endometrium Ki67 immunohistochemical staining showed a large number of brownish yellow nuclei, which were distributed evenly. In the uterus wound repair process, cells in surrounding normal tissues migrate, adhere and proliferate to the damaged area, so that the cell content of the damaged area is increased, and the tissue repair is promoted. After 4 weeks, only a small amount of proliferating cells in the endometrium of the control group are distributed, the proliferating cells in the test group are more than those in the control group, and a large amount of proliferating cells can be seen in the endometrium and are uniformly distributed in the whole endometrium. After 8 weeks, the number of the proliferation cells in the test group with the reconstruction of the endometrium and the muscular layer is still higher than that of the control group, and is similar to that of the normal endometrium.

Statistical results of the number of proliferating cells (Ki67) in the surgical field showed (fig. 11): 4 weeks after surgery, the number of proliferating cells/field of the test group (400 ×) was: 90.33 +/-9.29, is higher than that of a sham operation group by 47.13 +/-6.77 and is obviously higher than that of a control group by 45.75 +/-8.95, and the comparison difference has statistical significance. After 8 weeks, the number of the proliferation cells in the uterine operation area of each group is reduced compared with the previous group, but the number of the proliferation cells in the test group is still higher and is close to that in the sham operation group. The number of proliferating cells/field of the test group (400 ×) was: 53.20 + -3.63, the difference was statistically significant compared to the control group 15.60 + -3.11.

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 many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. An implant for inducing uterine wall tissue regeneration in situ, which is characterized in that the interior of the implant has a three-dimensional nano-net structure and is made of raw materials containing a fibrinogen compound and a polylactic acid polycaprolactone copolymer.

2. An implant as claimed in claim 1, characterised in that 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;

more preferably, the fibrinogen is porcine blood-derived fibrinogen.

3. The implant of claim 1, wherein the mass ratio of the fibrinogen complex to the polylactic acid-polycaprolactone copolymer is (0.48-1.1): 1.

Preferably, the molecular weight of the polylactic acid polycaprolactone copolymer is 5-30 ten thousand.

4. The implant of claim 1, wherein the protein content of the implant is 100-220 mg/g; residual protein <12 mg/g.

5. The implant of claim 1, wherein the implant is in the form of a membrane;

preferably, the thickness of the implant is between 0.51mm and 1.4mm, more preferably between 0.6mm and 0.8 mm.

6. Implant according to claim 1, characterized in that the porosity of the implant is between 41% and 80%, preferably between 45% and 70%;

and/or the water absorption of the implant is 35-200%, preferably 55-80%;

and/or the tensile strength of the implant is 0.5-5.0 MPa, preferably 1.0-4.0 MPa;

and/or the elongation at break of the implant is 60% to 200%, preferably 70% to 160%.

7. An implant according to any of claims 1 to 6, wherein the implant is produced by electrospinning.

8. The implant according to claim 7, wherein the implant is prepared by uniformly mixing a solution containing the fibrinogen complex and a solution containing the polylactic acid-polycaprolactone copolymer, adding the mixture into the same volumetric tube of an electrospinning machine, and electrospinning; or respectively adding the solution containing the fibrinogen compound and the solution containing the polylactic acid-polycaprolactone copolymer into two different volumetric tubes of an electrostatic spinning machine, and simultaneously carrying out electrostatic spinning preparation;

preferably, the solution containing the polylactic acid polycaprolactone copolymer is prepared by dissolving the polylactic acid polycaprolactone copolymer in a mixed solvent of one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran and acetone at a mass volume percentage concentration of 5-8%;

preferably, the solution containing the fibrinogen complex is prepared by dissolving the fibrinogen complex in distilled water at a mass volume percentage concentration of 8.0-29.0%;

preferably, the voltage difference of the electrostatic spinning is 15-140 Kv, and/or the electrospinning distance is 10-50 cm, and/or the electrospinning liquid advancing speed is 3-399mL/h and 401-960 mL/h.

9. Method for implanting an implant according to any of claims 1 to 8, comprising the steps of: the implant is placed between the muscular layers of the uterine wall or between the serosal layer and the muscular layers of the uterine wall.

10. Use of an implant according to any one of claims 1 to 8 in the preparation of a material for repairing a defect in a body tissue.

11. Use according to claim 10, wherein the material is a repair material for the treatment of meninges, abdominal defects, pelvic floor organ prolapse, atria, ventricular septum, pericardial defects, tendon or ligament rupture, or parenchymal organ rupture.

12. Use of an implant according to any one of claims 1 to 8 in the manufacture of a material for inducing regeneration of uterine wall tissue in situ or for preventing scar tissue formation or for preventing and treating complications following uterine surgery;

preferably, the uterine wall tissue comprises serosa, uterine smooth muscle, endometrium, connective tissue and extracellular matrix;

preferably, the complications include pelvic adhesions, endometriosis, uterine rupture upon re-pregnancy, pre-placenta, placenta implantation upon re-pregnancy, long term adverse reactions such as pregnancy at the scar site of a uterine incision from caesarean section.

13. Use of a hydrophilic electrospun biological composite scaffold material in the preparation of a material for in situ induction of uterine wall tissue regeneration or prevention of scar tissue formation or prevention and treatment of uterine postoperative complications, characterized in that the composite scaffold material is prepared by blending fibrinogen, L-arginine or an aqueous solution of hydrochloride thereof with a P (LLA-CL) solution, using an electrospinning technique; wherein the mass ratio of the fibrinogen to the L-arginine or the hydrochloride thereof is 1.2: 1-12.5: 1;

14. The use according to claim 13, wherein the uterine wall tissue comprises serosa, uterine smooth muscle, endometrium, connective tissue and extracellular matrix.

15. Use according to claim 13, characterized in that said complications include pelvic adhesions, endometriosis, uterine rupture in the event of a second pregnancy, implantation of a pre-placenta, placenta in the event of a second pregnancy, distant adverse reactions such as pregnancy at the scar zone of a uterine incision from caesarean section.

16. Use according to any one of claims 13 to 15, wherein the fibrinogen is of mammalian origin.

17. The use according to claim 16, wherein the mammal is a human, pig, cow, sheep or horse.

18. Use according to any one of claims 13 to 15, wherein the mass ratio of polylactic acid to polycaprolactone in P (LLA-CL) is 20:80 to 95: 5.

19. Use according to any one of claims 13-15, wherein the solvent in the P (LLA-CL) solution is selected from one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran, chloroform or acetone.

20. The use according to any one of claims 13 to 15, 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 its hydrochloride with the solution of P (LLA-CL).

21. Use according to any one of claims 13 to 15, wherein the hydrophilic electrospun bio-composite scaffold material has an equilibrium contact angle of less than 55 °.

22. The use of any one of claims 13-15, wherein the hydrophilic electrospun bio-composite scaffold material has a total volume shrinkage after contact with an aqueous solution of no more than 20%; the porosity is not less than 30%.

23. Use according to any one of claims 13 to 15, wherein 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, lincosamines, fosfomycin, chloramphenicol, colistin B, bacitracin.

24. Use according to claim 23, 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.

25. Use according to claim 23, 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.