CN116196478A

CN116196478A - Rotator cuff patch containing exosomes and preparation method thereof

Info

Publication number: CN116196478A
Application number: CN202310077942.9A
Authority: CN
Inventors: 石锐; 薛芸; 姜春岩
Original assignee: BEIJING RESEARCH INSTITUTE OF TRAUMATOLOGY AND ORTHOPAEDICS; Beijing Jishuitan Hospital
Current assignee: BEIJING RESEARCH INSTITUTE OF TRAUMATOLOGY AND ORTHOPAEDICS; Beijing Jishuitan Hospital
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2023-06-02

Abstract

The invention discloses a rotator cuff patch containing an exosome and a preparation method thereof, wherein a coaxial electrostatic spinning method is adopted to prepare nanofibers, so that the ultra-microstructure of an extracellular matrix is simulated, and meanwhile, the nanofiber structure is similar to rotator cuff tissue, so that rotator cuff tissue can be simulated; and the nanofiber has enough mechanical strength and elasticity, can guide the tissue to grow in a proper direction, and has ideal biomedical functions. The core layer contains exosomes, can promote migration and fusion of osteoblasts and tendon cells, and has the effect of promoting tissue healing; meanwhile, as the exosomes are encapsulated in the nanofiber core layer structure and release is slower than that of common blend nanofibers, the core-shell structure can slow down the exosomes release and avoid burst release.

Description

Rotator cuff patch containing exosomes and preparation method thereof

Technical Field

The invention belongs to the technical field of bone tissue material preparation, and particularly relates to a rotator cuff patch containing exosomes and a preparation method thereof.

Background

The rotator cuff is a sleeve-like structure formed by tendons of supraspinatus, subglottis, small circular muscle and subscapularis surrounding the humeral head, which stabilize the humeral head on the glenoid, play an important role in the stabilization of the shoulder joint while maintaining the movement of the shoulder joint, which, once damaged, can cause pain and dysfunction of the shoulder joint. Rotator cuff tear is one of the most common shoulder diseases, with rotator cuff tear rates of 34% for average persons and up to 54% for elderly persons over 60 years old. Despite improvements in surgical instruments and techniques, extensive rotator cuff tear remains a challenge for orthopedics. The current strategy for surgical treatment is to repair the torn rotator cuff tendon. However, the literature reports that the re-rupture rate after repair is 11% -94%. Although current clinical treatments such as arthroscopic surgery can improve patient prognosis by reducing pain and improving shoulder function, the rate of postoperative recurrent defects (re-tears) is still not ideal (21% of follow-up 1-2 years), and rotator cuff tendons play an important role in shoulder biomechanics, and are susceptible to injury and degenerative changes due to their location and blood supply. Even with tendon-bone attachment, the high tension of the repair structure increases the risk of re-tearing. Accordingly, grafts have been developed for bridging rotator cuff large-area tears. However, the limited healing potential between tendons and grafts or between grafts and bones remains a problem.

The electrostatic spinning nanofiber has wide application in the fields of tissue engineering, wound repair, drug delivery, nerve probe, orthopedic implants and the like. These nanofibers comprise nanofibers oriented in different directions to mimic the ultrastructure of the extracellular matrix, especially in tissues with complex areas, such as tendon-endosteal. The organization and arrangement of the nanofibers can be adjusted during the manufacturing process, which allows the structural and material properties of the scaffold to meet the functional requirements of the rotator cuff tendon. The aligned scaffolds mimic the parallel extracellular matrix of the natural cellular microenvironment, enhance matrix deposition, and act as a three-dimensional micropattern for new tissue formation, with non-aligned scaffolds or randomly oriented fibers having good results in osteogenesis and chondrogenesis. Among the various electrospun polymers, polycaprolactone (PCL) is approved by the U.S. food and drug administration as a component of biomaterial scaffolds due to its tunable biodegradability. As a major component of the tendon extracellular matrix, collagen I (Col I) promotes cell growth, migration and differentiation through interaction with growth factors, and can enhance the cytocompatibility of polymers.

Exosomes are nano-sized (30-150 nm diameter) extracellular vesicles naturally secreted by a variety of cell types, including endothelial cells, immune cells, and also Mesenchymal Stem Cells (MSCs) from different sources such as bone marrow, fat and human umbilical cord. The most attractive feature of exosomes is that they are cell-free, they are less complex from a regulatory point of view, and clinical trials are underway after emergency use authorization by the U.S. Food and Drug Administration (FDA) is obtained. The lipid bilayer structure of exosomes enables transfer of information encoded in proteins, lipids, mRNA, miRNA and metabolites, thereby facilitating intracellular communication. In contrast to MSCs, MSCs-derived exosomes exhibit similar biological activities, such as anti-inflammatory, pro-regenerative and anti-apoptotic effects. Thus, the therapeutic effects and mechanisms of exosomes in the tissue healing process have led to an increasing interest in enhancing rotator cuff repair.

However, currently clinically accepted and commercially available rotator cuff patches, such as X-Repair, LARS alignment, poly-Tape, etc., do not sufficiently approximate the macroscopic and microscopic mechanical properties of the supraspinatus tendon, exhibiting insufficient mechanical properties. In addition, the developed novel rotator cuff patch has the problem of biocompatibility such as biological stents, platelet-rich matrixes and other materials; when the untreated human body tissue graft material is obtained, secondary injury can be generated to the human body, and the tissue such as fat and the like possibly can be contained, so that tendon is difficult to grow.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the rotator cuff patch containing the exosome and the preparation method thereof, which can effectively solve the technical problems of insufficient mechanical property, biocompatibility and low biosafety of the existing rotator cuff patch.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses a rotator cuff patch containing an exosome, which is of a core-shell nanofiber membrane structure; wherein the core layer is made of exosomes and high-molecular polymers, and the shell layer is made of high-molecular polymers;

the raw materials for preparing the rotator cuff patch containing the exosomes comprise the following components in percentage by mass:

the core layer: 1-15% of high molecular polymer, 1-20% of exosome and 65-92% of phosphate buffer solution;

the shell layer is as follows: 5 to 30 percent of high molecular polymer and 70 to 95 percent of hexafluoroisopropanol.

Preferably, the high molecular polymer in the core layer adopts gelatin or polyvinyl alcohol; the high polymer in the shell layer adopts polycaprolactone, polylactic acid-glycolic acid or a mixture of polylactic acid-glycolic acid and collagen.

Further preferably, the polyvinyl alcohol has a viscosity of 30 to 220mpa.s; the number average molecular weight of the polycaprolactone is 10000-100000; the viscosity of the polylactic acid-glycolic acid is 0.5-3 dL/g; the mass ratio of the polylactic acid-glycolic acid to the collagen in the mixture of the polylactic acid-glycolic acid and the collagen is (5-9): (5-1).

Further preferably, the collagen is type I collagen.

Preferably, the exosomes are derived from cells or body fluids, including stem cells, macrophages, endothelial cells or epithelial cells.

The invention also discloses a preparation method of the rotator cuff patch containing the exosome, which comprises the following steps:

1) Dissolving the high molecular polymer for preparing the shell layer in hexafluoroisopropanol, and fully and uniformly mixing to prepare a shell layer spinning solution;

2) Adding the high molecular polymer for preparing the nuclear layer into phosphate buffer solution, heating and stirring for 5-10 h, and adding exosome after the temperature is restored to room temperature to prepare nuclear layer spinning solution;

3) And (3) carrying out coaxial electrostatic spinning treatment on the shell layer spinning solution and the core layer spinning solution, and spraying the coaxial electrostatic spinning treatment on an aluminum foil paper substrate to obtain the rotator cuff patch containing the exosome.

Preferably, in the step 1), the mixture is fully and uniformly mixed by magnetic stirring for 5-10 hours at room temperature.

Preferably, in the step 2), when the high molecular polymer adopts polyvinyl alcohol, the heating temperature is 80-100 ℃; when gelatin is used as the high molecular polymer, the heating temperature is 37 ℃.

Preferably, in step 2), the pH of the phosphate buffer is 7.4.

Preferably, in step 3), the operating parameters of the coaxial electrospinning process are: the propelling speed of the spinning solution of the shell layer is 1-2 mL/h, the propelling speed of the spinning solution of the core layer is 0.5-1 mL/h, the rotating speed of the roller is 350rpm, the voltage is set to be 15-20 KV, and the distance between the needle point and the collector is 15cm.

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

according to the preparation method of the rotator cuff patch containing the exosome, disclosed by the invention, the coaxial electrostatic spinning method is adopted to prepare the nanofiber, so that the ultrastructure of an extracellular matrix is simulated, and meanwhile, the nanofiber structure is similar to rotator cuff tissue, so that rotator cuff tissue can be simulated; and the nanofiber has enough mechanical strength and elasticity, can guide the tissue to grow in a proper direction, and has ideal biomedical functions. The coaxial electrostatic spinning is used for preparing the nanofiber with the core-shell structure, and the core layer contains an exosome, so that migration and fusion of osteoblasts and tendon cells can be promoted, and the nanofiber has the function of promoting tissue healing; meanwhile, the exosomes are encapsulated in the nanofiber core layer structure and are released more slowly than common blending nanofibers, so that the core-shell structure can slow down the exosomes release and avoid burst release.

The rotator cuff patch containing the exosomes prepared by the method is characterized in that collagen is an important constituent component of rotator cuff, PLGA provides enough mechanical strength and elasticity, the exosomes are rich in various proteins and miRNAs, participate in various biological processes and have a promoting effect on rotator cuff injury healing. Its advantages are mainly: 1. the patch has enough mechanical strength and elasticity to meet the requirement of the rotator cuff patch; the fiber bending direction can guide the tissue to grow to a proper direction, promote tissue repair and have ideal biomedical functions; 2. after tissue regeneration reaches the availability level, the patch can be completely degraded, and the biocompatibility is good; 3. the patch contains exosome component and can promote tissue regeneration.

Drawings

Fig. 1 is a TEM image of an exosome-containing rotator cuff patch according to example 1 of the present invention.

FIG. 2 is a macro-topography of an exosome-containing rotator cuff patch of inventive example 1

Fig. 3 is an SEM image of an exosome-containing rotator cuff patch of inventive example 1.

FIG. 4 is a graph showing the in vitro cell activity of the exosome-containing rotator cuff patch of inventive example 1.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

example 1

(1) Cell culture, exosome isolation and identification: bone marrow mesenchymal stem cells were cultured in an alpha-MEM medium (Hyclone) containing 100U/ml penicillin-0.1 mg/ml streptomycin antibiotic in an amount of 10% by volume of fetal bovine serum (Gibco). After growing to 60% of cell density, the culture medium was changed to 5% volume fraction exosome-free serum medium (SBI company), and the conventional culture was continued for 48 hours, and cell supernatant was collected; exosomes were isolated from the cell supernatants by ExoQuick-TC kit (System Bioscience). The isolated exosomes were resuspended in 1X PBS.

(2) Preparing a nanofiber membrane, namely a shoulder blade patch, and an inner layer by adopting an electrostatic spinning method: preparing a shell spinning solution, namely dissolving 1.4g of polylactic acid-glycolic acid copolymer (PLGA) with the viscosity of 1.8dL/g in 18g of hexafluoroisopropanol, adding 0.6g of collagen, and stirring for 5 hours by a magnetic stirrer at room temperature; preparing a core layer spinning solution: preparing exosome solutions with different concentrations: to 20g of PBS, 2g of polyvinyl alcohol (PVA) having a viscosity of 80-110 and mPs was added, and after stirring at 90℃for 5 hours, 0.1%, 1% and 10% of exosomes were added after the temperature was returned to room temperature. Carrying out electrostatic spinning on the two prepared solutions, wherein the advancing speed of the shell layer solution is 2mL/h, the advancing speed of the core layer solution is 1mL/h, the rotating speed of a roller is 350rpm, the voltage is set to 20KV, and the distance between a needle point and a collector is 15cm.

(3) Nanofiber configuration characterization: the electrospun nanofibers were received with a copper mesh placed on a collector and TEM characterized the core-shell structure of the nanofibers, indicating that the nanofibers had a core-shell structure (fig. 1). Referring to fig. 2 and 3, the exosome-containing rotator cuff patch prepared by the invention has complete overall shape, and the nanofiber composite rotator cuff tissue is bionic, so that the growth and arrangement of cells can be regulated.

(4) And (3) mechanical property detection: the mechanical properties of the nanofiber membranes were characterized by an electronic tensile tester, each membrane was cut into 5 samples of dimensions 4mm x 25mm, the test environment temperature was 25 ℃, and the tensile strength was measured. As shown in Table 1, the tensile strength of the exosome-containing coaxial electrospun nanofiber membrane was significantly greater than that of pure PLGA. The coaxial electrostatic spinning nanofiber membrane containing the exosome has higher tensile strength and can imitate the structure and functions of natural fibers of rotator cuff tissues.

TABLE 1

(5) Nanofiber membrane in vitro biocompatibility: and taking tenocytes as model cells, and testing the in-vitro biocompatibility of different types of nanofiber membranes. The viability of tenocytes proliferating on different types of nanofibrous membranes was assessed by CCK-8 assay. The membrane was cut into a circular shape of 3cm in diameter, sterilized, and then fixed in the wells of a 24-well plate by Cell-Crown (TM). Tenocyte in 3×10 ⁴ The individual cells/ml density was resuspended in DMEM medium supplemented with 10% FBS and antibiotics. Then, willA mixture of cell culture medium and cell suspension was inoculated onto the sample at 37℃with 5% CO ₂ The cells are cultured under conditions. Cell culture medium was changed every two days. On

days

1, 3, 5 and 7, 100. Mu.l of CCK-8 solution was added to the medium of each well and incubated for 2h. The medium was then transferred to a 96-well plate and OD was measured at 450 nm. The results are shown in FIG. 4, the cell viability of the exosome-containing nanofiber membrane on tenocytes gradually increased from day 1 to day 7, and the absorbance was highest compared with the other groups, indicating that the ability to promote cell proliferation was best.

Example 2

(1) Cell culture, exosome isolation and identification: umbilical cord mesenchymal stem cells were cultured in DMEM/F-12 medium (Beijing Soy Co., ltd.) supplemented with 10% fetal bovine serum (Gibco Co.) and 1% streptomycin antibiotic containing 100U/ml penicillin-0.1 mg/ml streptomycin. After growing to 60% of cell density, the culture medium was changed to 5% volume fraction exosome-free serum medium (SBI company), and the conventional culture was continued for 48 hours, and cell supernatant was collected; exosomes were isolated from the cell supernatants by ExoQuick-TC kit (System Bioscience). The isolated exosomes were resuspended in 1X PBS.

(3) And (3) mechanical property detection: the mechanical properties of the nanofiber membranes were characterized by an electronic tensile tester, each membrane was cut into 5 samples of dimensions 4mm x 25mm, the test environment temperature was 25 ℃, and the tensile strength was measured. As shown in Table 2, the tensile strength of the exosome-containing coaxial electrospun nanofiber membrane was significantly greater than that of pure PLGA. The coaxial electrostatic spinning nanofiber membrane containing the exosome has higher tensile strength and can imitate the structure and functions of natural fibers of rotator cuff tissues.

TABLE 2

As can be seen from comparison with example 1, compared with the marrow mesenchymal stem cell-derived exosome nanofiber membrane, the umbilical cord mesenchymal stem cell-derived exosome nanofiber membrane has similar mechanical properties, which indicates that the two exosomes from different sources have little influence on the mechanical properties.

Example 3

(2) Preparing a nanofiber membrane, namely a shoulder blade patch, and an inner layer by adopting an electrostatic spinning method: preparing a shell spinning solution, namely dissolving 3g of polylactic acid-glycolic acid copolymer (PLGA) with the viscosity of 3dL/g in 14g of hexafluoroisopropanol, adding 3g of collagen, and stirring for 5 hours by a magnetic stirrer at room temperature; preparing a core layer spinning solution: preparing exosome solutions with different concentrations: 4g of polyvinyl alcohol (PVA) with the viscosity of 30-80mPs is added into 16g of PBS, the mixture is heated to 90 ℃ and stirred for 5 hours, and 15% of exosomes are added after the temperature is restored to room temperature. Carrying out electrostatic spinning on the two prepared solutions, wherein the advancing speed of the shell layer solution is 2mL/h, the advancing speed of the core layer solution is 1mL/h, the rotating speed of a roller is 400rpm, the voltage is set to 20KV, and the distance between a needle point and a collector is 15cm.

(3) And (3) mechanical property detection: the mechanical properties of the nanofiber membranes were characterized by an electronic tensile tester, each membrane was cut into 5 samples of dimensions 4mm x 25mm, the test environment temperature was 25 ℃, and the tensile strength was measured. As shown in Table 3, the tensile strength of the exosome-containing coaxial electrospun nanofiber membrane was significantly greater than that of pure PLGA. The coaxial electrostatic spinning nanofiber membrane containing the exosome has higher tensile strength and can imitate the structure and functions of natural fibers of rotator cuff tissues.

TABLE 3 Table 3

Example 4

(1) Preparing a shell spinning solution, namely dissolving 2g of Polycaprolactone (PCL) in 18g of hexafluoroisopropanol, and stirring for 5 hours by a magnetic stirrer at room temperature;

(2) Preparing a core layer spinning solution: preparing exosome solutions with different concentrations: to 18g of PBS, 2g of gelatin was added, and after stirring at 37℃for 5 hours, 0.1%, 1% and 10% of exosomes were added after the temperature was returned to room temperature.

(3) And preparing the nanofiber membrane by adopting an electrostatic spinning method. Carrying out electrostatic spinning on the two prepared solutions, wherein the advancing speed of the shell layer solution is 2mL/h, the advancing speed of the core layer solution is 1mL/h, the rotating speed of a roller is 350rpm, the voltage is set to 20KV, and the distance between a needle point and a collector is 15cm.

(4) And (3) mechanical property detection: the mechanical properties of the nanofiber membranes were characterized by an electronic tensile tester, each membrane was cut into 5 samples of dimensions 4mm x 25mm, the test environment temperature was 25 ℃, and the tensile strength was measured. As shown in Table 4, the tensile strength of the exosome-containing coaxial electrospun nanofiber membrane was significantly greater than that of pure PCL. The coaxial electrostatic spinning nanofiber membrane containing the exosome has higher tensile strength and can imitate the structure and functions of natural fibers of rotator cuff tissues.

TABLE 4 Table 4

Example 5

(1) Preparing a shell spinning solution, namely dissolving 6g of Polycaprolactone (PCL) in 14mL of hexafluoroisopropanol, and stirring for 5 hours by a magnetic stirrer at room temperature;

(2) Preparing a core layer spinning solution: preparing exosome solutions with different concentrations: to 19.9g of PBS, 0.1g of gelatin was added, and the mixture was stirred at 37℃for 5 hours, and after the temperature was returned to room temperature, 1% of exosomes were added.

(4) And (3) mechanical property detection: the mechanical properties of the nanofiber membranes were characterized by an electronic tensile tester, each membrane was cut into 5 samples of dimensions 4mm x 25mm, the test environment temperature was 25 ℃, and the tensile strength was measured. As shown in Table 5, the tensile strength of the exosome-containing coaxial electrospun nanofiber membrane was significantly greater than that of pure PCL. The coaxial electrostatic spinning nanofiber membrane containing the exosome has higher tensile strength and can imitate the structure and functions of natural fibers of rotator cuff tissues.

TABLE 5

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. The rotator cuff patch containing the exosome is characterized in that the rotator cuff patch is of a core-shell nanofiber membrane structure; wherein the core layer is made of exosomes and high-molecular polymers, and the shell layer is made of high-molecular polymers;

2. The exosome-containing rotator cuff patch of claim 1, wherein the high molecular polymer in the core layer is gelatin or polyvinyl alcohol; the high polymer in the shell layer adopts polycaprolactone, polylactic acid-glycolic acid or a mixture of polylactic acid-glycolic acid and collagen.

3. An exosome-containing rotator cuff patch according to claim 2 wherein the polyvinyl alcohol has a viscosity of 30 to 220mpa.s; the number average molecular weight of the polycaprolactone is 10000-100000; the viscosity of the polylactic acid-glycolic acid is 0.5-3 dL/g; the mass ratio of the polylactic acid-glycolic acid to the collagen in the mixture of the polylactic acid-glycolic acid and the collagen is (5-9): (5-1).

4. An exosome-containing rotator cuff patch according to claim 2 wherein the collagen is type I collagen.

5. The exosome-containing rotator cuff patch of claim 1, wherein the exosome is derived from a cell or body fluid, the cell comprising a stem cell, a macrophage, an endothelial cell, or an epithelial cell.

6. A method of preparing an exosome-containing rotator cuff patch according to any one of claims 1 to 5 comprising the steps of:

7. The method of claim 6, wherein in step 1), the mixing is performed by magnetic stirring at room temperature for 5-10 hours.

8. The method for preparing an exosome-containing rotator cuff patch according to claim 6, wherein in step 2), when the high molecular polymer is polyvinyl alcohol, the heating temperature is 80-100 ℃; when gelatin is used as the high molecular polymer, the heating temperature is 37 ℃.

9. The method of claim 4, wherein in step 2), the phosphate buffer solution has a pH of 7.4.

10. The method of preparing an exosome-containing rotator cuff patch according to claim 4, wherein in step 3), the operating parameters of the coaxial electrospinning process are: the propelling speed of the spinning solution of the shell layer is 1-2 mL/h, the propelling speed of the spinning solution of the core layer is 0.5-1 mL/h, the rotating speed of the roller is 350rpm, the voltage is set to be 15-20 KV, and the distance between the needle point and the collector is 15cm.