An acromioclavicular joint prosthesis and its preparation method and application

Abstract

The present invention discloses a rotator cuff patch, its preparation method and application, belonging to the technical field of composite biomaterials in medical engineering. The present invention prepares an oriented nanofiber patch by electrospinning a synthetic biodegradable high polymer and a natural bioactive protein of biological origin. Calcium silicate nanoparticles are in-situ coprecipitated at the distal end of the oriented nanofiber patch, and connective tissue growth factor is loaded at the proximal end of the oriented nanofiber patch to obtain the rotator cuff patch. It is a gradient composite rotator cuff patch with a tendon-promoting region loaded with connective tissue growth factor, a cartilage-promoting region co-loaded with connective tissue growth factor and calcium silicate, and a bone-promoting region with calcium silicate deposition. It can be used for the treatment of acute rotator cuff tears, effectively realizing multi-tissue regeneration of tendon, cartilage and bone, and then synchronously completing tendon-bone interface healing and promoting rotator cuff injury repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of composite biomaterials in biomedical engineering, and particularly relates to a rotator cuff patch, a preparation method thereof, and an application thereof. Background Art

[0002] Rotator cuff tear is a relatively common musculoskeletal disease, which usually causes severe pain in the patient's shoulder and limited functional activities. Surgical repair of the damaged rotator cuff site through arthroscopic technology is an effective means for treating rotator cuff tear. However, the incidence of retear after rotator cuff repair surgery is 26.6%-94.0%, and the retear rate is still relatively high. The regenerative ability of the tendon-to-bone gradient component is limited, and the mechanical properties of the tendon-bone interface recovered after surgery are far worse than those of the normal fibrocartilaginous tendon-bone healing structure. Eventually, it will lead to poor healing of the tendon-bone interface and retear, and the generation of a large stress concentration at the tendon-bone interface is considered to be the main cause of retear after rotator cuff repair. The patch can serve as a mechanical support structure during rotator cuff repair to disperse the stress concentration at the interface. Using a patch to enhance the fixation between the tendon and the bone can prevent retear after rotator cuff repair, and it is currently considered a promising treatment strategy.

[0003] However, the existing rotator cuff patches still have the following problems: First, the mechanical properties of the patch cannot adapt to the regenerative needs of natural tissues. The mechanical properties of the patch play an important role in the healing of the tendon-bone interface. If the mechanical properties of the patch are insufficient, it may fail prematurely; if the elasticity of the patch is insufficient, it may hinder tissue growth due to stress shielding. When the shoulder joint moves, the patch will stretch along with the tendon. Therefore, the patch needs to have good tensile load to withstand the tensile force brought by tendon movement and prevent premature fracture and failure due to insufficient mechanical properties. The patch also needs to have good elasticity to conform to the dynamic process of tendon stretching and rebound during tendon stretching and avoid hindering the normal movement of the rotator cuff. Second, the bioactivity and functionality of the patch are insufficient. The bioactivity and functionality of the patch material used to repair rotator cuff injuries are key factors for successful repair of the tendon-bone interface. The patch material first needs to have good in vivo biocompatibility to ensure the adhesion, survival, and proliferation of endogenous cells on its surface. Rotator cuff patches with poor activity may cause some complications, such as infection, rejection reaction, etc. The lack of biological functionality often manifests as insufficient promotion of multi-directional differentiation of cells by the patch, which will affect the synchronous regeneration of multiple tissues at the tendon-bone interface and ultimately limit the healing effect of the damaged site after rotator cuff repair.

[0004] Therefore, it is necessary to provide a rotator cuff patch with biomimetic mechanical properties and gradient-induced regeneration of the tendon-bone interface. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a rotator cuff patch, a preparation method thereof and an application. The rotator cuff patch is sutured and fixed at the damaged tendon-bone interface. One end of the rotator cuff patch is sutured to the supraspinatus muscle, defined as the proximal end of the patch; the other end is fixed to the humerus, defined as the distal end of the patch. The rotator cuff patch of the present invention is a gradient-structured rotator cuff patch loaded with connective tissue growth factor and calcium silicate, and an oriented nanofiber patch co-spun from a synthetic biodegradable polymer and a natural bioactive protein of biological origin is prepared by electrospinning. Calcium silicate nanoparticles are in-situ co-precipitated at the distal end of the patch, and connective tissue growth factor is loaded at the proximal end of the patch. Through the hydrogen bond connection between the protein and the connective tissue growth factor, a gradient composite rotator cuff patch with a tendon-promoting area loaded with connective tissue growth factor, a cartilage-promoting area co-loaded with connective tissue growth factor and calcium silicate, and a bone-promoting area with calcium silicate deposition is finally prepared.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] The present invention provides a preparation method of a rotator cuff patch. An oriented nanofiber patch co-spun from a synthetic biodegradable polymer and a natural bioactive protein of biological origin is prepared by electrospinning. Calcium silicate nanoparticles are in-situ co-precipitated at the distal end of the oriented nanofiber patch, and connective tissue growth factor is loaded at the proximal end of the oriented nanofiber patch to obtain the rotator cuff patch.

[0009] The rotator cuff patch is sutured and fixed at the damaged tendon-bone interface. One end of the rotator cuff patch is sutured to the supraspinatus muscle, defined as the proximal end of the patch; the other end is fixed to the humerus, defined as the distal end of the patch.

[0010] Connective tissue growth factor (CTGF) was first discovered by BRADHAM et al. in 1991 in the conditioned medium of human umbilical vein endothelial cells. It is a cysteine-rich secreted peptide composed of 349 amino acids with a molecular weight of 34-38 KD.

[0011] Further, the preparation method of the rotator cuff patch includes the following steps:

[0012] S1. Using a synthetic biodegradable polymer and a natural bioactive protein of biological origin as raw materials, and hexafluoroisopropanol as a solvent, an oriented nanofiber patch is prepared by electrospinning. Since the nanofiber arrangement in the present invention is regular in direction rather than randomly distributed, and is similar to the parallel arrangement of tendon fibers, it is called an "oriented nanofiber patch".

[0013] S2. Vertically immerse the distal end of the aligned nanofiber patch in a calcium nitrate tetrahydrate solution, freeze-dry after the reaction. In this step, calcium ions form coordination bonds with the carbonyl and carboxyl groups in the natural bioactive proteins of biological origin and bind to the surface of the nanofiber patch.

[0014] S3. Vertically immerse the distal end of the nanofiber patch obtained in S2 in a sodium silicate nonahydrate solution, freeze-dry after the reaction to prepare a gradient patch with calcium silicate loaded at the distal end. In this step, silicate ions combine with calcium ions, and calcium silicate nanoparticles are in-situ deposited on the surface of the nanofibers (the reaction equation is: Ca(NO3)2 + Na2SiO3 → CaSiO3↓ + 2NaNO3). Due to capillary action and the gravity of the solution, more calcium silicate is deposited on the patch near the distal end, while relatively less calcium silicate is distributed on the patch near the proximal end. Therefore, a gradient patch with calcium silicate loaded at the distal end is obtained.

[0015] S4. Vertically immerse the proximal end of the gradient patch prepared in S3 in a connective tissue growth factor solution, freeze-dry after the reaction to load connective tissue growth factor at the proximal end of the gradient patch and obtain the rotator cuff patch. In this step, the proximal end of the gradient patch is vertically immersed in the connective tissue growth factor solution for reaction. Hydrogen bonds are formed between the connective tissue growth factor protein and the natural bioactive proteins of biological origin. Due to capillary action and the gravity of the solution, more connective tissue growth factor is adsorbed on the patch near the proximal end, while relatively less connective tissue growth factor is adsorbed on the patch near the distal end. Finally, a gradient-structured rotator cuff patch loaded with connective tissue growth factor and calcium silicate is obtained.

[0016] In the method of the present invention, a synthetic biodegradable polymer and a natural bioactive protein of biological origin are used as raw materials, and hexafluoroisopropanol is used as a solvent. After mixing, they are stirred for 6 - 10 h to obtain an electrospinning solution.

[0017] Exemplarily, the synthetic biodegradable polymer includes polycaprolactone, a copolymer of lactide and caprolactone, or a copolymer of lactide and glycolide; and / or

[0018] The natural bioactive protein of biological origin includes at least one of collagen, keratin, fibrin, and elastin, preferably type I collagen derived from bovine Achilles tendon.

[0019] Furthermore, the mass ratio of the synthetic biodegradable polymer to the natural bioactive protein of biological origin is (8 - 13.5)∶(1 - 3). For example, when the mass of the synthetic biodegradable polymer is 1.35 g, the mass of the natural bioactive protein of biological origin is 0.15 g.

[0020] Further, the mass-volume ratio of the synthetic biodegradable polymer to hexafluoroisopropanol is (0.8 - 1.35) g : 10 mL.

[0021] Further, during the electrospinning, the flow rate of the spinning solution is 2 mL / h, the working voltage is 14 - 16 kV, the distance between the syringe needle and the aluminum foil receiver is 14 - 16 cm, and the rotational speed of the aluminum foil receiver is 1500 - 2000 r / min. Preferably, the volume of the spinning solution is 4 mL, the flow rate of the spinning solution is 2 mL / h, the working voltage is 15 kV, the distance between the syringe needle and the aluminum foil receiver is 15 cm, and the rotational speed of the aluminum foil receiver is 1500 r / min.

[0022] After the electrospinning is completed, the prepared oriented nanofiber patch is placed in a vacuum dryer for drying treatment for 48 h to fully remove the residual solvent on the fiber patch.

[0023] Further, the concentration of the calcium nitrate tetrahydrate solution is 10 - 40 mmol / L. The reaction time in the calcium nitrate tetrahydrate solution is 2 h, and the freeze-drying time after the reaction is completed is 24 h.

[0024] Further, the concentration of the sodium silicate nonahydrate solution is 10 - 40 mmol / L. The reaction time in the sodium silicate nonahydrate solution is 2 h, and the freeze-drying time after the reaction is completed is 24 h.

[0025] Further, the concentration of the connective tissue growth factor solution is 100 - 200 ng / mL. The soaking time in the connective tissue growth factor solution is 22 - 26 h, and the freeze-drying time after the reaction is completed is 24 h.

[0026] The principle of the present invention is:

[0027] 1. Complementarity between the synthetic biodegradable polymer and the natural bioactive protein of biological origin: The synthetic biodegradable polymer has good mechanical properties, showing characteristics of high toughness and low elasticity, while some natural bioactive proteins of biological origin have good elasticity and the property of being able to quickly return to their original state after being subjected to external forces. During the electrospinning process, the synthetic biodegradable polymer and the natural bioactive protein of biological origin will interact with each other, such as forming hydrogen bonds, van der Waals forces, etc. These interactions help to enhance the binding force between the fibers, resulting in a significant improvement in the mechanical properties of the rotator cuff patch.

[0028] 2. Physicochemical properties of the patch surface: Synthetic biodegradable polymers usually exhibit strong hydrophobicity. A hydrophobic surface easily causes protein denaturation, which is not conducive to cell adhesion and growth. After binding with natural bioactive proteins of biological origin, the hydrophilicity of the patch is enhanced, and the cell affinity of the patch is increased. The hydrophilic surface can adsorb more adhesion proteins, which provide adhesion sites for cells, help maintain the conformation and activity of adhesion proteins, and thus enhance the interaction with the material surface.

[0029] The second technical solution of the present invention:

[0030] The present invention also provides a rotator cuff patch prepared by the above method, which is a gradient structure rotator cuff patch loaded with connective tissue growth factor and calcium silicate.

[0031] The third technical solution of the present invention:

[0032] The present invention also provides the application of the rotator cuff patch in the preparation of a repair material for tendon-bone interface injury.

[0033] Compared with the prior art, the present invention has the following advantages and technical effects:

[0034] 1. In terms of preparation process: The raw materials of the patch prepared by the present invention are synthetic biodegradable polymers and natural bioactive proteins of biological origin respectively. Synthetic biodegradable polymers (such as polycaprolactone) are commonly used biomedical materials that have been approved by the US FDA and are usually used as in vivo implant materials and drug controlled release materials; natural bioactive proteins of biological origin (such as collagen or keratin) all have good biocompatibility and biodegradable safety, and have good immunogenicity. The present invention prepares the patch material by electrospinning, which has the characteristics of simple preparation process, mild preparation conditions, no toxic substances such as chemical crosslinking agents, and the prepared product is non-toxic and has high biocompatibility.

[0035] 2. In terms of material properties: The patch prepared by the present invention uses a polymer material as the main raw material, and the prepared patch has good mechanical properties. After adding a certain proportion of collagen or keratin, the mechanical properties of the patch are further significantly improved, changing the characteristics of high toughness and low elasticity of the polymer material. The prepared patch has better performance in terms of elastic modulus and tensile strength, can meet the mechanical load required during rotator cuff tendon repair, and effectively plays a role in stress dispersion during the repair process.

[0036] 3. In terms of product function: The rotator cuff patch prepared by the present invention is a multifunctional patch. By releasing two active substances, it realizes the functions of inducing multi-directional differentiation of cells and promoting the repair and regeneration of multiple tissues. Connective tissue growth factor and calcium silicate, which have significant promoting effects on the repair of tendon tissue and bone tissue respectively, are selected. And the synergistic effect of connective tissue growth factor and calcium silicate can more significantly promote the chondrogenic differentiation of cells. The loading of the two active substances effectively promotes the repair of three gradient tissues, which is better than the repair effect achieved by loading three active substances in the past. It can be used for the treatment of acute rotator cuff tears, effectively realizing the multi-tissue regeneration of tendon, cartilage and bone, and then synchronously completing the healing of the tendon-bone interface and promoting the repair of rotator cuff injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0038] Figure 1 It is the mechanical property diagram of the rotator cuff patch in Example 1 of the present invention;

[0039] Figure 2 It is the in-vivo effect diagram of promoting the healing of the rotator cuff tendon-bone interface of the rotator cuff patch in Example 1 of the present invention;

[0040] Figure 3 It is the in-vivo effect diagram of promoting the regeneration of fibrocartilage at the rotator cuff tendon-bone interface of the rotator cuff patch in Example 1 of the present invention;

[0041] Figure 4 It is the in-vivo effect diagram of promoting the bone regeneration at the rotator cuff tendon-bone interface of the rotator cuff patch in Example 1 of the present invention. A shows the reconstruction and regeneration of the newly formed bone at the tendon-bone interface at 4 weeks and 8 weeks. B shows the quantitative results of the bone volume fraction at 4 weeks and 8 weeks. C shows the quantitative results of the bone density at 4 weeks and 8 weeks;

[0042] In Figure 2 and Figure 3 , B represents Bone (bone). When discussing the rotator cuff tendon-bone interface, bone is an important component because it involves the connection between tendon and bone; I represents Interface (interface). In this case, it refers to the connection interface between tendon and bone (including uncalcified fibrocartilage and calcified fibrocartilage), which is a key area because it involves the combination of two different types of tissues; T represents Tendon (tendon). The rotator cuff tendon is a group of tendons of the shoulder rotator muscle group. They connect muscles to bones and play an important role in shoulder movement. DETAILED DESCRIPTION OF THE INVENTION

[0043] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0044] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0046] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0047] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0048] Unless otherwise specified, the normal temperature in the present invention is calculated as 25 ± 2°C.

[0049] Each raw material used in the embodiments of the present invention is obtained by purchasing commercially.

[0050] In the present invention, the rotator cuff patch is sutured and fixed to the tendon-bone interface. One end of the rotator cuff patch that contacts the supraspinatus tendon region is defined as the proximal end, and one end of the rotator cuff patch that contacts the humerus region is defined as the distal end.

[0051] In some embodiments of the present invention, a method for preparing a rotator cuff patch is provided, including the following steps:

[0052] S1. Using a synthetic biodegradable polymer and a natural bioactive protein of biological origin as raw materials, and hexafluoroisopropanol (HFIP) as a solvent, after mixing and stirring for 6 - 10 h, an electrospinning solution is obtained. An oriented nanofiber patch is prepared by electrospinning. The mass ratio of the synthetic biodegradable polymer to the natural bioactive protein of biological origin is (8 - 13.5):(1 - 3), and the preferred mass ratio is 9:1. For example, when the mass of the synthetic biodegradable polymer is 1.35 g, the mass of the natural bioactive protein of biological origin is 0.15 g; the mass - volume ratio of the synthetic biodegradable polymer to hexafluoroisopropanol is (0.8 - 1.35) g:10 mL. During electrospinning, when the volume of the spinning solution is 4 mL, the flow rate of the spinning solution is 2 mL / h, the working voltage is 15 kV, the distance between the syringe needle and the aluminum foil receiver is 15 cm, and the rotational speed of the aluminum foil receiver is 1500 r / min. After electrospinning, the prepared oriented nanofiber patch is placed in a vacuum dryer for drying treatment for 48 h to fully remove the residual solvent on the fiber patch;

[0053] S2. Vertically immerse the distal end of the nanofiber patch in a calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) solution with a concentration of 10 - 40 mmol / L for 2 h, and then freeze - dry for 24 h. In this step, calcium ions form coordination bonds with the carbonyl and carboxyl groups in the natural bioactive protein of biological origin and bind to the surface of the nanofiber patch;

[0054] S3. Vertically immerse the distal end of the nanofiber patch obtained in S2 in a sodium silicate nonahydrate (Na2SiO3·9H2O) solution with a concentration of 10 - 40 mmol / L for 2 h, and then freeze - dry for 24 h to obtain a gradient patch with calcium silicate (CS) loaded at the distal end. In this step, silicate ions combine with calcium ions, and calcium silicate nanoparticles are in - situ deposited on the surface of the nanofibers (the reaction equation is: Ca(NO3)2+Na2SiO3→CaSiO3↓+2NaNO3). Through capillary action and the gravitational action of the solution, more calcium silicate is deposited on the patch near the distal end, while relatively less calcium silicate is distributed on the patch near the proximal end. Therefore, a gradient patch with calcium silicate loaded at the distal end is obtained;

[0055] S4. Vertically immerse the proximal end of the gradient patch prepared in S3 into a connective tissue growth factor (CTGF) solution with a concentration of 100 - 200 ng / mL. After reacting for 22 - 26 h, freeze-dry for 24 h to load connective tissue growth factor at the proximal end of the gradient patch, obtaining the rotator cuff patch. In this step, by vertically immersing the proximal end of the gradient patch into the connective tissue growth factor solution for reaction, hydrogen bonds are formed between the connective tissue growth factor protein and the natural bioactive protein of biological origin. Through capillary action and the gravitational force of the solution, the patch closer to the proximal end adsorbs more connective tissue growth factor, while the patch closer to the distal end adsorbs relatively less connective tissue growth factor, ultimately obtaining a gradient-structured rotator cuff patch loaded with connective tissue growth factor and calcium silicate.

[0056] In some embodiments of the present invention, the synthetic biodegradable polymer includes polycaprolactone (PCL), a copolymer of lactide and caprolactone, or a copolymer of lactide and glycolide. Preferably, the copolymer of lactide and caprolactone is poly-L-lactide-caprolactone (PLCL), and more preferably PCL; the natural bioactive protein of biological origin includes at least one of collagen, keratin, fibrin, and elastin. For example, the natural bioactive protein of biological origin is type I collagen (COLⅠ) derived from bovine Achilles tendon, keratin (KRT) derived from wool, and preferably COLⅠ. The synthetic biodegradable polymer has good mechanical properties, showing characteristics of high toughness and low elasticity, while some natural bioactive proteins of biological origin have good elasticity and the property of being able to quickly return to their original state after being subjected to external forces. The synthetic biodegradable polymer and the natural bioactive protein of biological origin will interact during the electrospinning process, such as forming hydrogen bonds, van der Waals forces, etc. These interactions help to enhance the binding force between the fibers, resulting in a significant improvement in the mechanical properties of the composite fibers (oriented nanofiber patch). In the following typical embodiments of the present invention, PCL, PLCL, COLⅠ, and KRT are taken as examples for illustration.

[0057] The abbreviations involved in the following examples specifically refer to:

[0058] PCL: Polycaprolactone;

[0059] PLCL: Poly-L-lactide-caprolactone;

[0060] COLⅠ: Type I collagen from bovine Achilles tendon;

[0061] KRT: Keratin from wool

[0062] HFIP: Hexafluoroisopropanol;

[0063] CS: Calcium silicate;

[0064] CTGF: Connective tissue growth factor;

[0065] PC: PCL / COLⅠ;

[0066] PLC: PLCL / COLⅠ;

[0067] PK: PCL / KRT;

[0068] PLK: PLCL / KRT;

[0069] PC@CTGF / CS: A PCL / COLⅠ patch with calcium silicate loaded at the distal end and connective tissue growth factor loaded at the proximal end.

[0070] PLC@CTGF / CS: A PLCL / COLⅠ patch with calcium silicate loaded at the distal end and connective tissue growth factor loaded at the proximal end.

[0071] PK@CTGF / CS: A PCL / KRT patch with calcium silicate loaded at the distal end and connective tissue growth factor loaded at the proximal end.

[0072] PLK@CTGF / CS: A PLCL / KRT patch with calcium silicate loaded at the distal end and connective tissue growth factor loaded at the proximal end.

[0073] It should be noted that the parts not described in detail in the present invention are all conventional operation means in the art and are not the focus of the present invention.

[0074] The technical solution of the present invention will be further described below through examples.

[0075] Example 1

[0076] A preparation method of a rotator cuff patch with connective tissue growth factor and calcium silicate loaded at both ends:

[0077] 1) Prepare the PC electrospinning solution:

[0078] Dissolve 1.35 g of PCL and 0.15 g of COLⅠ in 10 mL of HFIP, stir well for 8 h to obtain a 15 wt% PC solution, and the mass ratio of PCL:COLⅠ is 9:1.

[0079] 2) Prepare the PC electrospun patch:

[0080] Take 4 mL of the PC solution, set the flow rate of the spinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 15 cm, and the rotation speed of the aluminum foil receiver to 1500 r / min. After electrospinning, obtain the PC patch, and place the obtained PC patch in a vacuum dryer for drying treatment for 48 h to fully remove the residual solvent on the PC patch.

[0081] 3) Preparation of PC@CTGF / CS composite patch:

[0082] Place the distal end of the PC patch obtained in step 2) in a 10 mmol Ca(NO3)2·4H2O solution, soak it under vacuum conditions for 2 h, treat it in a vacuum dryer for 24 h, soak it in a 10 mmol Na2SiO3·9H2O solution under vacuum conditions for 2 h, and treat it in a vacuum dryer for 24 h to obtain a PC patch with CS loaded at the distal end; place the proximal end of the PC patch with CS loaded at the distal end in a 100 ng / mL CTGF solution, soak it at 4 °C for 24 h, and treat it in a freeze dryer for 24 h to finally obtain the PC@CTGF / CS composite patch.

[0083] Example 2

[0084] A method for preparing a rotator cuff patch with connective tissue growth factor and calcium silicate loaded at both ends:

[0085] 1) Preparation of PLC electrospinning solution:

[0086] Dissolve 1.35 g of PLCL and 0.15 g of COLⅠ in 10 mL of HFIP, stir well for 8 h to obtain a 15 wt% PLC solution, with a mass ratio of PLCL:COLⅠ of 9:1.

[0087] 2) Preparation of PLC electrospinning patch:

[0088] Take 4 mL of the PLC solution, set the flow rate of the spinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 15 cm, and the rotation speed of the aluminum foil receiver to 1500 r / min. After electrospinning, obtain the PLC patch, and place the obtained PLC patch in a vacuum dryer for 48 h to fully remove the residual solvent on the PLC patch.

[0089] 3) Preparation of PLC@CTGF / CS composite electrospun patch:

[0090] Place the distal end of the PLC patch obtained in step 2) in 10 mmol Ca(NO3)2·4H2O, soak it under vacuum conditions for 2 h, treat it in a vacuum dryer for 24 h, soak it in 10 mmol Na2SiO3·9H2O under vacuum conditions for 2 h, and treat it in a vacuum dryer for 24 h to obtain a PLC patch with CS loaded at the distal end. Place the proximal end of the PLC patch with CS loaded at the distal end in a 100 ng / mL CTGF solution, soak it at 4 °C for 24 h, and treat it in a freeze dryer for 24 h to finally obtain the PLC@CTGF / CS composite patch.

[0091] Example 3

[0092] A preparation method of a rotator cuff patch with dual - end loading of connective tissue growth factor and calcium silicate:

[0093] 1) Prepare the PK electrospinning solution:

[0094] Dissolve 1.35 g of PCL and 0.3 g of KRT in 10 mL of HFIP, and stir well for 8 h to obtain a 16.5 wt% PK solution.

[0095] 2) Prepare the PK electrospun patch:

[0096] Take 4 mL of the PK solution, set the flow rate of the spinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 15 cm, and the rotation speed of the aluminum foil receiver to 1500 r / min. After electrospinning, obtain the PK patch, and place the obtained PK patch in a vacuum dryer for 48 h to fully remove the residual solvent on the PK patch.

[0097] 3) Prepare the PK@CTGF / CS composite electrospun patch:

[0098] Place the distal end of the PK patch obtained in step 2) in 40 mmol of Ca(NO3)2·4H2O, soak it under vacuum conditions for 2 h, treat it in a vacuum dryer for 24 h, then soak it in 40 mmol of Na2SiO3·9H2O under vacuum conditions for 2 h, and treat it in a vacuum dryer for 24 h to obtain a PK patch with CS loaded at the distal end. Place the proximal end of the PK patch in a 200 ng / mL CTGF solution, soak it at 4 °C for 24 h, and treat it in a freeze dryer for 24 h to finally obtain the PK@CTGF / CS composite patch.

[0099] Example 4

[0100] A preparation method of a rotator cuff patch with dual - end loading of connective tissue growth factor and calcium silicate:

[0101] 1) Prepare the PLK electrospinning solution:

[0102] Dissolve 0.8 g of PLCL and 0.1 g of KRT in 10 mL of HFIP, and stir well for 8 h to obtain a 9 wt% PLK solution, with PLCL:KRT being 8:1 (mass ratio).

[0103] 2) Prepare the PLK electrospun patch:

[0104] Take 4 mL of the PLK solution, set the flow rate of the spinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 15 cm, and the rotation speed of the aluminum foil receiver to 1500 r / min. Place the obtained PLK patch in a vacuum dryer for 48 h to fully remove the residual solvent on the patch.

[0105] 3) Preparation of PLK@CTGF / CS composite electrospun patch:

[0106] Place the distal end of the PLK patch obtained in step 2) in 20 mmol Ca(NO3)2·4H2O, soak it under vacuum conditions for 2 h, treat it in a vacuum dryer for 24 h, soak it in 20 mmol Na2SiO3·9H2O under vacuum conditions for 2 h, and treat it in a vacuum dryer for 24 h to obtain a PLK film with CS loaded at the distal end. Place the proximal end of the PLK patch in a CTGF solution of 100 ng / mL, soak it at 4 °C for 24 h, and treat it in a freeze dryer for 24 h to finally obtain the PLK@CTGF / CS composite patch.

[0107] After testing, the properties of the patches prepared in the above Examples 1-4 are similar and there are no significant differences. Only the rotator cuff patch prepared in Example 1 will be used as an example to illustrate the properties below.

[0108] I. Patch mechanical property experiment

[0109] The mechanical properties were evaluated by a universal material testing machine. Each sample was cut into a rectangular strip with a length of 30 mm, a width of 10 mm, and a thickness of 0.2 mm. The tensile experiment was carried out at room temperature with a tensile speed of 10 mm / min. The tensile specimen was stretched until tensile fracture, and the elastic modulus, ultimate load, tensile strength, and elongation at break were recorded and calculated. The rotator cuff tendon-bone interface repeatedly bears mechanical loads in daily life. Therefore, tensile properties and elasticity are essential attributes of the patch in the repair application of rotator cuff injuries. The mechanical experiment results of the PC patch are as Figure 1 shown. The average elastic modulus of the PC patch is 46.35 ± 4.81 MPa, which is 3.98 times that of the PCL patch; the average tensile strength is 23.59 ± 1.88 MPa, which is 4.77 times that of the PCL patch. The PC patch has better mechanical properties, is sufficient to match the load of the natural supraspinatus tendon, and can meet the clinical application requirements.

[0110] II. Healing effect of tendon-bone interface

[0111] An acute rotator cuff tear model was established in rats. A longitudinal skin incision was made on the anterolateral side of the shoulder joint, and the deltoid muscle was incised to expose the supraspinatus tendon, whose insertion point was attached to the greater tubercle of the humerus. The supraspinatus muscle was separated from the insertion with a No. 10 scalpel, and the exposed tendon-bone attachment point was gently dissected to ensure complete removal of the supraspinatus tendon attachment. Two parallel bone tunnels were established from the tendon-bone attachment point to the lateral side of the humeral head using a 0.5-mm diameter Kirschner wire. The end of the supraspinatus tendon was passed through the bone tunnel with a 3-0 suture, and the suture was tied to the humeral cortex. The corresponding patch was sutured above the tendon-bone repair site to form a "double-layer structure", with the proximal end of the patch fixed to the supraspinatus tendon and the distal end fixed to the humerus. The rotator cuff area of the Normal group was not treated; the direct suture group was named the Suture group; the group repaired with the PC patch (product of step 2 of Example 1) was named the PC group; the group repaired with the PC@CTGF / CS composite patch was named the PC@CTGF / CS group. Samples were taken after 4 weeks and 8 weeks of in vivo repair for histological staining (H&E staining, safranin O-fast green staining) and Micro-CT to verify the repair effect of the patch of the present invention on the rotator cuff tendon-bone interface.

[0112] The hematoxylin and eosin staining results of the tendon-bone interface are as Figure 2 shown. At 4 weeks in the initial stage of repair, the distribution of the tendon-bone interface in each group was discontinuous, different from natural tissue, and obvious inflammatory cell infiltration occurred in the Suture group, while the inflammatory infiltration in the PC group and the PC@CTGF / CS group was relatively mild. As the repair time extended to 8 weeks, the tendon-bone interface in each group healed further, and the inflammatory infiltration in each group was significantly reduced. The tendon-bone interface in the PC@CTGF / CS group healed best, with no obvious boundary between tissues, and the morphology of the tendon-bone interface after healing was closer to natural tissue.

[0113] III. Fibrocartilage regeneration effect

[0114] The safranin-fast green staining results are as Figure 3 shown. The dark red-stained area at the tendon-bone interface refers to extracellular matrix proteoglycan. It is significantly reflected that the production and deposition of glycosaminoglycan in the PC@CTGF / CS group are more than those in other groups at different repair nodes, and the fibrocartilage regeneration in the PC@CTGF / CS group is better. The above results indicate that the patch prepared in the present invention loaded with connective tissue growth factor and calcium silicate at both ends effectively promotes the fibrocartilage regeneration of the tendon-bone interface.

[0115] IV. New bone formation effect

[0116] The reconstruction and regeneration of new bone at the tendon-bone interface were observed by Micro-CT. As Figure 4As shown in A, at 4 weeks and 8 weeks, the bone defect area in the Suture group was larger. At 8 weeks, bone regeneration in the PC group was better, but the new bone surface of the humerus was not smooth. Compared with the Suture group and the PC group, the new bone regeneration in the PC@CTGF / CS group was better and was most similar to the normal group. There was no obvious difference between the new bone tissue in the PC@CTGF / CS group and the surrounding normal tissue, and complete regeneration was basically achieved at 8 weeks. Figure 1 B and C in the figure are the quantitative results of bone volume fraction (BV / TV) and bone mineral density (BMD) at 4 weeks and 8 weeks respectively. At 8 weeks, the BMD and BV / TV in the PC@CTGF / CS group were 1.49 times and 1.14 times that of the Suture group respectively. The above results all indicate that the PC@CTGF / CS patch can significantly promote bone regeneration at the tendon-bone interface.

[0117] In summary, the present invention successfully prepared a rotator cuff patch PC@CTGF / CS with dual-terminal loading of connective tissue growth factor and calcium silicate. This patch can cover the damaged rotator cuff tendon-bone interface, form a bilayer structure with the tendon-bone attachment point, play a stress dispersion role in the damaged area, and reduce the stress concentration at the tendon-bone interface during the repair process; by releasing the loaded connective tissue growth factor and calcium silicate in a regional manner, it can effectively promote the regeneration of tendon, cartilage, and bone tissues, synchronously achieve the healing of the tendon-bone interface, and is beneficial to the repair of the rotator cuff tendon-bone interface.

[0118] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for preparing a rotator cuff patch, characterized in that: The following steps are involved: S1. Using synthetic biodegradable polymers and natural bioactive proteins from biological sources as raw materials and hexafluoroisopropanol as solvent, oriented nanofiber patches were prepared by electrospinning; S2. vertically immersing the distal end of the oriented nanofiber patch in a calcium nitrate tetrahydrate solution, and freeze-drying after the reaction; S3. The distal end of the nanofiber patch obtained in S2 is vertically immersed in a sodium silicate nonahydrate solution, and freeze-dried after the reaction to obtain a gradient patch loaded with calcium silicate at the distal end; S4. Vertically immerse the proximal end of the gradient patch prepared in S3 in a connective tissue growth factor solution, freeze-dry after the reaction, load the connective tissue growth factor on the proximal end of the gradient patch, and obtain the rotator cuff patch.

2. The method for preparing a rotator cuff patch according to claim 1, characterized in that: The synthetic biodegradable polymer includes polycaprolactone, a copolymer of lactide and caprolactone or a copolymer of lactide and glycolide; and / or The biologically derived natural bioactive protein includes at least one of collagen, keratin, fibrin and elastin.

3. The method for preparing a rotator cuff patch according to claim 2, characterized in that: The mass ratio of the synthetic biodegradable polymer to the natural biologically active protein of biological origin is (8-13.5): (1-3).

4. The method for preparing a rotator cuff patch according to claim 1, characterized in that: During the electrospinning, the flow rate of the spinning solution is 2 mL / h, the working voltage is 14-16 kV, the distance between the syringe needle and the aluminum foil receiver is 14-16 cm, and the rotation speed of the aluminum foil receiver is 1500-2000 r / min.

5. The method for preparing a rotator cuff patch according to claim 1, characterized in that: The concentration of the calcium nitrate tetrahydrate solution is 10-40 mmol / L.

6. The method for preparing a rotator cuff patch according to claim 1, characterized in that: The concentration of the sodium silicate nonahydrate solution is 10-40 mmol / L.

7. The method for preparing a rotator cuff patch according to claim 1, characterized in that: The connective tissue growth factor solution has a concentration of 100-200 ng / mL.

8. A rotator cuff patch, characterized in that: Prepared according to the method according to any one of claims 1 to 7.

9. Use of the rotator cuff patch according to claim 8 in preparing tendon-bone interface damage repair material.

Images