Acellular matrix composite rotator cuff patch

Abstract

The present application provides an acellular matrix composite rotator cuff patch, which includes an acellular dermis layer and an acellular pericardium layer, wherein the acellular dermis layer is an upper layer and the acellular pericardium layer is a lower layer. The acellular matrix composite rotator cuff patch provided in the present application has a simple preparation method, and the acellular matrix composite rotator cuff patch has good mechanical properties, biocompatibility, and tendon-bone healing performance, can reduce the postoperative re-tear rate, promote tendon-bone healing, and improve the success rate of rotator cuff injury repair surgery, and has good application prospects.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to an acellular matrix composite rotator cuff patch. Background Art

[0002] Rotator cuff tears are a common tendon injury in orthopedics and sports medicine, and are a common cause of shoulder pain, decreased range of motion, and reduced function. The incidence of rotator cuff tears in the general population is 20.7%, and the tear rate increases with age. The re-tear rate after rotator cuff repair is 20%-30%, reaching 40%-50% for massive rotator cuff tears. The overall repair failure rate is as high as 94%, making it a current clinical challenge and a key issue.

[0003] Patches are considered to be ideal for tension-free or micro-tension sutures. Foreign homologous / heterogeneous rotator cuff patches are mainly derived from the dermis, pericardium, or small intestinal submucosa (SIS). After a series of treatments, only extracellular matrix components such as collagen and elastic fibers are retained, while cells and active protein components that cause tissue rejection reactions are completely removed. However, no rotator cuff patch products have been approved for marketing in China, and there is still a gap. Therefore, there is an urgent need for a rotator cuff patch that can promote tendon-bone healing and reduce the rate of postoperative retear, improve the postoperative rehabilitation effect of patients after rotator cuff repair, and open up new ways for doctors to treat rotator cuff tears. Summary of the Invention

[0004] The purpose of this application is to provide an acellular matrix composite rotator cuff patch to reduce the rate of postoperative retear, promote tendon-bone healing, and improve the success rate of rotator cuff repair surgery. The specific technical solution is as follows:

[0005] The present application provides an acellular matrix composite rotator cuff patch, which includes an acellular dermis layer and an acellular pericardium layer, wherein the acellular dermis layer is an upper layer and the acellular pericardium layer is a lower layer.

[0006] The acellular matrix composite rotator cuff patch provided in this application has a simple preparation method. The acellular matrix composite rotator cuff patch has good mechanical properties, biocompatibility and tendon-bone healing performance, can reduce the postoperative re-tear rate, promote tendon-bone healing, and improve the success rate of rotator cuff injury repair surgery, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0008] Figure 1 The tensile strength results of the rotator cuff patches prepared in Example 1 and Comparative Examples 1-3 are shown;

[0009] Figure 2 The suture strength results of the rotator cuff patches prepared in Example 1 and Comparative Examples 1-3 are shown;

[0010] Figure 3 The cytotoxicity test results of the rotator cuff patches prepared in Example 1 and Comparative Examples 1-3 are shown;

[0011] Figure 4 The figures show the pathological results of repairing chronic massive rotator cuff tears in rabbits using the rotator cuff patches prepared in Example 1 and Comparative Examples 1-3. DETAILED DESCRIPTION

[0012] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field based on this application are within the scope of protection of this application.

[0013] The present application provides an acellular matrix composite rotator cuff patch, which includes an acellular dermis layer and an acellular pericardium layer, wherein the acellular dermis layer is an upper layer and the acellular pericardium layer is a lower layer.

[0014] The acellular matrix composite rotator cuff patch provided in the present application combines the acellular dermis layer and the acellular pericardium layer to form a double-layer structure, using the acellular dermis layer as a mechanical support material and the acellular pericardium layer as a material to promote tendon-bone healing, so that it can promote tendon-bone healing while reducing the postoperative re-tear rate; wherein, the acellular matrix composite rotator cuff patch is implanted in the recipient site in the form of a scaffold, and the three-dimensional structure, collagen, non-collagenous protein and growth factor components it contains provide a suitable environment for host cell adhesion, proliferation and differentiation, thereby promoting the regeneration of collagen fibers and blood vessels, and contributing to the functional reconstruction of tissues such as bone, cartilage and tendon, thereby promoting tendon-bone healing after rotator cuff repair.

[0015] In some embodiments of the present application, the thickness of the acellular dermis layer is 1-2.5 mm.

[0016] In some embodiments of the present application, the acellular dermis layer consists of 1-2 layers of acellular dermis, and the acellular dermis is prepared by the following method:

[0017] Cross-linking: Dermal tissue was immersed in a cross-linking solution with a pH of 5-6 containing 10-50 mM (mmol / L) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 10-50 mM N-hydroxysuccinimide (NHS), and 10-100 mM 2-(N-morpholino)ethanesulfonic acid (MES). The solution was shaken at room temperature for 3-24 hours and then washed with purified water to obtain a cross-linked intermediate.

[0018] Decellularization: taking the cross-linked intermediate, soaking it in a decellularization solution containing 0.5-1M (mol / L) NaOH, shaking it at room temperature for 4-24 hours, washing it with phosphate buffered saline (PBS), and then washing it with purified water to obtain the decellularized dermis;

[0019] Wherein, the dermal tissue is selected from the back dermis of humans, pigs, cows or fetal cows.

[0020] In the present application, the solvent of the cross-linking solution is water; the decellularization solution containing 0.5-1 M NaOH is an aqueous solution containing 0.5-1 M NaOH.

[0021] In the present application, cross-linking treatment can enhance the mechanical properties of the acellular dermis and reduce the re-tear rate after surgery; it can also block the surface antigens of the acellular dermis, thereby reducing the immune response after implantation in the body and improving the biocompatibility of the material.

[0022] In some embodiments of the present application, the thickness of the decellularized pericardium layer is 0.2-1 mm.

[0023] In some embodiments of the present application, the decellularized pericardium layer consists of 2-5 layers of decellularized pericardium, and the thickness of the decellularized pericardium is 0.1-0.5 mm.

[0024] In the present application, the increase in the number of decellularized pericardium layers increases its matrix content, thereby promoting cell proliferation and further promoting tendon-bone healing.

[0025] In the present application, the surface of the decellularized pericardium is divided into a dense surface and a loose surface, which correspond to the fibrous layer and the serous wall layer, respectively. The loose surface has a porous structure, which is conducive to cell infiltration and rapid vascularization, thereby improving the performance of promoting tendon-bone healing.

[0026] In the present application, the decellularized pericardium layer can be formed by stacking the decellularized pericardium by physical bonding methods such as suturing or negative pressure, and the loose surface of the pericardium is controlled to face outwards during stacking (ie, it can be close to the affected area when used).

[0027] In some embodiments of the present application, the decellularized pericardium is prepared by the following method:

[0028] Decellularization: The pericardial tissue was collected and immersed in a decellularization solution containing 0.1-1 M NaOH, shaken at room temperature for 10-120 minutes, washed with PBS, and then washed with purified water to obtain the decellularized pericardium;

[0029] Wherein, the pericardial tissue is selected from human, pig, cow or fetal bovine.

[0030] In the present application, the decellularization solution containing 0.1-1 M NaOH is an aqueous solution containing 0.1-1 M NaOH.

[0031] The acellular matrix composite rotator cuff patch provided in the present application has acellular dermis that has been cross-linked and decellularized as the upper layer for mechanical support, and acellular pericardium that has been decellularized as the lower layer to promote tendon-bone healing.

[0032] In the present application, the PBS is a PBS buffer commonly used in the art, and those skilled in the art can prepare it according to the existing formula. The PBS buffer usually includes Na2HPO4, KH2PO4, NaCl and KCl; illustratively, the preparation of the PBS buffer includes: weighing NaCl, KCl, Na2HPO4, KH2PO4 and dissolving them in water, adjusting the solution to pH = 7.3-7.5 with HCl, and finally adding water to make up the volume to obtain a PBS buffer, wherein the ratio of NaCl mass, KCl mass, Na2HPO4 mass, KH2PO4 mass, and water volume is 7-9g:0.1-0.3g:1.4-1.5g:0.2-0.3g:1L.

[0033] The following examples and comparative examples are provided to further illustrate the embodiments of the present application, but the present application is not limited to the following examples. The experimental materials and methods used in the following examples are conventional materials and methods unless otherwise specified. Preparation of PBS: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 were weighed and dissolved in 800 mL of distilled water. The solution was adjusted to pH 7.4 with HCl, and the volume was finally made up to 1 L with distilled water to obtain PBS buffer.

[0034] Test method:

[0035] Tensile strength test:

[0036] According to GB / T 1040.2-2022 Plastics - Determination of tensile properties - Part 2: Test conditions for molded and extruded plastics, the test was carried out using a 5A dumbbell specimen at a tensile speed of 10 mm / min.

[0037] Suture strength test:

[0038] The test was carried out in accordance with 5.1.5 Suture strength of "YY / T 1814-2022 Surgical Implants Synthetic Non-absorbable Mesh Hernia Repair Mesh".

[0039] Cytotoxicity test:

[0040] In accordance with GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests and GB / T 16886.12-2017 Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials, the test was carried out using the extract method and L929 cells.

[0041] Animal experiments:

[0042] A chronic massive rotator cuff tear model was established by artificially resecting the supraspinatus tendon of New Zealand rabbits for 6 weeks. The massive rotator cuff tear was repaired using a double-row suture technique and a patch. The repair progress was observed at 16 weeks. The tendon-bone interface was stained with hematoxylin and eosin (HE) and Masson's trichrome, and the supraspinatus muscle was stained with HE for microscopic observation.

[0043] Example 1

[0044] <Preparation of acellular dermis>

[0045] Cross-linking: Porcine dorsal dermis was immersed in a cross-linking solution containing 30 mM EDC, 30 mM NHS, 50 mM MES, pH 5.5, shaken at room temperature for 16 hours, and washed with purified water to obtain a cross-linked intermediate.

[0046] Decellularization: The cross-linked intermediate was immersed in a decellularization solution containing 1M NaOH, shaken at room temperature for 4 hours, washed with PBS until neutral, and then washed with purified water to obtain acellular dermis with a thickness of 1.2 mm.

[0047] <Preparation of Decellularized Pericardium>

[0048] Decellularization: Porcine pericardium tissue was obtained and immersed in a decellularization solution containing 0.1 M NaOH, shaken at room temperature for 1 hour, washed with PBS, and then washed with purified water to obtain a decellularized pericardium with a thickness of 0.3 mm.

[0049] <Preparation of acellular matrix composite rotator cuff patch>

[0050] One layer of acellular dermis and two layers of acellular pericardium were sutured to obtain an acellular matrix composite rotator cuff patch.

[0051] Comparative Example 1

[0052] <Preparation of acellular dermis>

[0053] Decellularization: Porcine dorsal dermis was immersed in a decellularization solution containing 1 M NaOH, shaken at room temperature for 4 hours, washed with PBS until neutral, and then washed with purified water to obtain acellular dermis with a thickness of 1.2 mm.

[0054] <Preparation of Decellularized Pericardium>

[0055] Same as Example 1;

[0056] <Preparation of acellular matrix composite rotator cuff patch>

[0057] One layer of acellular dermis and two layers of acellular pericardium were sutured to obtain a rotator cuff patch.

[0058] Comparative Example 2

[0059] Dermal tissue from the pig's back was obtained, and acellular dermis was prepared according to the dermal tissue cross-linking and decellularization method in Example 1. Two layers of acellular dermis were sutured to obtain a rotator cuff patch.

[0060] Comparative Example 3

[0061] Porcine pericardial tissue was obtained, and decellularized pericardium was prepared according to the pericardial tissue decellularization method in Example 1. Four layers of decellularized pericardium were sutured to obtain a rotator cuff patch.

[0062] The rotator cuff patches prepared in Example 1 and Comparative Examples 1-3 were tested according to the above test method to obtain the tensile strength, suture strength and cytotoxicity test results of each rotator cuff patch. Figure 1 、 Figure 2 、 Figure 3 As shown, the results show that the tensile strength and suture strength of the rotator cuff patch of Example 1 are equivalent to those of Comparative Example 2, and are significantly higher than those of Comparative Example 1 and Comparative Example 3 (*P < 0.05, **P < 0.01), indicating that the acellular matrix composite rotator cuff patch of the present application has good mechanical properties; there is no significant difference in the cytotoxicity results of each rotator cuff patch, which shows that the acellular matrix composite rotator cuff patch of the present application has good biocompatibility.

[0063] The rotator cuff patches prepared in Example 1 and Comparative Examples 1-3 were subjected to the above animal experiments to obtain HE staining and Masson staining results of the tendon-bone interface and HE staining results of the supraspinatus muscle. Figure 4As shown, it can be seen that the tendon-bone interface of Comparative Examples 1 and 2 is clearly demarcated, with no good integration and regeneration, and the supraspinatus muscle tends to be fatty. The tendon-bone interface of Comparative Example 3 is richly vascularized and regenerates well, but the supraspinatus muscle is severely fatty. In contrast, the tendon-bone interface of Example 1 is excellent in regeneration and the degree of fatty formation of the supraspinatus muscle is minimal. The postoperative re-tear rate is related to the mechanical properties of the patch and the fatty formation of the muscle. Therefore, the tendon-bone interface regeneration performance of the acellular matrix composite rotator cuff patch of the present application is the best, and the possibility of re-tear after surgery is the lowest.

[0064] In summary, the acellular matrix composite rotator cuff patch provided in this application has good mechanical properties and biocompatibility, can effectively regenerate tendon-bone interface tissue, and reduce the postoperative re-tear rate.

[0065] The above description is only a preferred embodiment of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application are included in the scope of protection of the present application.

Claims

1. An acellular matrix composite rotator cuff patch, comprising an acellular dermis layer and an acellular pericardium layer, wherein the acellular dermis layer is an upper layer and the acellular pericardium layer is a lower layer, the acellular dermis layer has a thickness of 1-2.5 mm, and the acellular dermis layer is composed of 1-2 layers of acellular dermis, and the acellular dermis is prepared by the following method: Cross-linking: Dermal tissue was immersed in a cross-linking solution with a pH of 5-6 containing 10-50 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 10-50 mM N-hydroxysuccinimide, and 10-100 mM 2-(N-morpholino)ethanesulfonic acid. The solution was shaken at room temperature for 3-24 hours and then washed with purified water to obtain a cross-linked intermediate. Decellularization: taking the cross-linked intermediate, soaking it in a decellularization solution containing 0.5-1M NaOH, shaking it at room temperature for 4-24 hours, washing it with PBS, and then washing it with purified water to obtain the decellularized dermis; in, The dermal tissue is selected from the dorsal dermis of humans, pigs or cows.

2. The acellular matrix composite rotator cuff patch according to claim 1, wherein: The cattle are fetal cattle.

3. The acellular matrix composite rotator cuff patch according to claim 1 or 2, wherein: The thickness of the decellularized pericardium layer is 0.2-1 mm.

4. The acellular matrix composite rotator cuff patch according to claim 3, wherein: The decellularized pericardium layer consists of 2-5 layers of decellularized pericardium, and the thickness of the decellularized pericardium is 0.1-0.5 mm.

5. The acellular matrix composite rotator cuff patch according to claim 4, wherein: The decellularized pericardium is prepared by the following method: Decellularization: The pericardial tissue was collected and immersed in a decellularization solution containing 0.1-1 M NaOH, shaken at room temperature for 10-120 minutes, washed with PBS, and then washed with purified water to obtain the decellularized pericardium; Wherein, the pericardial tissue is selected from human, pig or cow.

6. The acellular matrix composite rotator cuff patch according to claim 5, wherein: The cattle are fetal cattle.

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