Rotator cuff tear shoulder arthritis model animal and rotator cuff tear shoulder arthritis model animal

Abstract

To provide a rotator cuff tear shoulder arthritis model animal and a method of producing a rotator cuff tear shoulder arthritis model animal, capable of more faithfully reproducing the pathology of rotator cuff tear shoulder arthritis.SOLUTION: A rotator cuff tear shoulder arthritis model animal is a rodent in which the supraspinatus, infraspinatus, superior joint capsule, and biceps longus tendons in the ipsilateral glenohumeral joint have been dissected.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an animal model of shoulder arthropathy caused by rotator cuff tear and a method for producing an animal model of shoulder arthropathy caused by rotator cuff tear. [Background technology]

[0002] A shoulder rotator cuff tear is the main cause of unbearable shoulder pain. More than 60% of people over 80 years old suffer from a shoulder rotator cuff tear. Some cases of shoulder rotator cuff tear progress to cuff tear arthropathy (hereinafter referred to as "CTA"), which is characterized by significant deformation of the shoulder joint (scapulohumeral joint), subchondral bone depression of the humerus, and bone loss at the humeral head. CTA causes severe shoulder pain and significantly restricts daily living activities by restricting the range of motion of the shoulder joint. CTA reduces the quality of life, such as making it impossible to live independently. CTA shortens the healthy life expectancy of the elderly. In addition, shoulder rotator cuff tears and CTA cause significant impairments in occupational and sports activities in young people as well. CTA places a large burden on society in terms of medical economy, as well as the physical and economic burden on patients.

[0003] The treatment of CTA is limited to reverse total shoulder arthroplasty. Therefore, elucidation of the pathology of CTA is expected in order to develop a treatment to prevent the onset of CTA after rotator cuff tear or to suppress the worsening of mild CTA. Primary osteoarthritis (OA) without rotator cuff tear is a deformity characterized by articular cartilage degeneration and osteophyte formation. OA is clearly distinguished from CTA both radiologically and histologically, and the molecular pathology of the two is expected to be different. Therefore, to elucidate the pathology of CTA, an animal model that accurately reproduces the characteristics of CTA is necessary, as existing knee joint OA models cannot be substituted.

[0004] Non-Patent Document 1 reports a CTA model rat in which the supraspinatus and infraspinatus muscles are cut, and Non-Patent Document 2 reports a CTA model mouse in which the supraspinatus, infraspinatus, and teres minor muscles are cut. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Erik J Kramer, 6 others, "Evaluation of cartilage degeneration in a rat model of rotator cuff tear arthropathy", J Shoulder Elbow Surg, 2013, 22(12): 1702-1709 [Non-Patent Document 2] Alissa Zingman, 8 others, “Shoulder arthritis secondary to rotator cuff tear: A reproducible murine model and histopathologic scoring system”, J Orthop Res, 2017, 35(3):506-514 Summary of the Invention [Problem to be solved by the invention]

[0006] In the CTA model rat reported in Non-Patent Document 1, it takes 12 weeks (equivalent to 8 years in a human lifetime) for the pathology of CTA to appear after surgery, and Non-Patent Document 1 does not mention the subchondral bone depression of the humerus joint or the bone loss of the humeral head. When the CTA model rat was reproduced, natural repair does not occur in the human rotator cuff, whereas in the CTA model rat, natural repair occurs by scar tissue. In the CTA model mouse reported in Non-Patent Document 2, it takes 45 weeks (equivalent to 36 years in a human lifetime) for the pathology of CTA to appear after surgery, so changes due to aging cannot be denied, and Non-Patent Document 2 does not mention the subchondral bone depression of the humerus joint. Furthermore, since tears of the teres minor muscle are rare in human rotator cuff tears, the CTA model mouse does not accurately reflect the pathology of CTA. Both the CTA model rat reported in Non-Patent Document 1 and the CTA model mouse reported in Non-Patent Document 2 show small changes related to the CTA phenotype, and it is difficult to say that they reflect the clinical practice of CTA.

[0007] The present invention has been made in consideration of the above-mentioned situation, and aims to provide a rotator cuff tear shoulder arthropathy model animal that can more faithfully reproduce the pathology of rotator cuff tear shoulder arthropathy and a method for producing a rotator cuff tear shoulder arthropathy model animal. [Means for solving the problem]

[0008] (Animal model of rotator cuff tear shoulder arthropathy) The rotator cuff tear shoulder arthropathy model animal described herein is A rodent in which the supraspinatus muscle tendon, the infraspinatus muscle tendon, the superior joint capsule, and the long head of the biceps brachii tendon at the same side of the glenohumeral joint are severed.

[0009] The rodent comprises: It may be a rat.

[0010] The rat may be 12 weeks of age or older.

[0011] The rotator cuff tear shoulder arthropathy model animal described in the present specification is The subchondral bone may be or be in a depressed state.

[0012] (Method for producing an animal model of rotator cuff tear shoulder arthropathy) The method for producing a model animal of rotator cuff tear shoulder arthropathy described in the present specification includes: The supraspinatus and infraspinatus tendons were dissected at the glenohumeral joint on the same side of the rodent. The upper joint capsule of the glenohumeral joint is dissected, The tendon of the long head of the biceps brachii is severed at the glenohumeral joint. Effect of the Invention

[0013] According to the present invention, the pathology of rotator cuff tear shoulder arthropathy can be reproduced more faithfully. [Brief description of the drawings]

[0014] [Figure 1] FIG. 2 is a schematic diagram showing an example of a site to be dissected in a model animal of shoulder arthropathy caused by rotator cuff tear according to an embodiment of the present invention. [Diagram 2] FIG. 2 shows images taken in chronological order from A to D of the surgical procedure for producing an improved rotator cuff tear arthropathy model in an example. [Diagram 3] 1 shows the results of evaluating glenohumeral joint cartilage degeneration in a conventional rotator cuff avulsion model. A shows an image of a hematoxylin-eosin stained section of the glenohumeral joint and surrounding tissue. B shows an image of an Alcian blue stained section of the glenohumeral joint and surrounding tissue. C shows a score that is an index of the degree of OA. [Figure 4] FIG. 13 is a photograph showing an image of the humeral head of an improved rotator cuff tear arthropathy model after 4 weeks in an example. [Diagram 5] FIG. 1 shows images of sections of the glenohumeral joint and surrounding tissue stained with Alcian blue in an improved rotator cuff tear arthropathy model in an example. [Figure 6] FIG. 1 shows a score, which is an index of the degree of OA in an improved rotator cuff tear arthropathy model in an example. [Figure 7]FIG. 13 shows images of sections of the glenohumeral joint and surrounding tissue stained with Safranin O after 4 weeks in an improved rotator cuff tear arthropathy model in an example. [Figure 8] FIG. 13 shows an image of a section of humeral head articular cartilage after 4 weeks from an improved rotator cuff tear arthropathy model in an example, obtained by immunohistochemical staining. [Figure 9] This is a diagram showing the results of bone histomorphometry of an improved rotator cuff tear arthropathy model in an embodiment. A shows a section of the subchondral bone of the humeral head stained with toluidine blue after 4 weeks. B shows the bone mass in the subchondral bone after 4 weeks. C shows the number of osteoclasts per unit area. D shows the mineralization rate. E shows the bone formation rate. [Figure 10] FIG. 13 shows an image of a section of the subchondral bone of the humeral head in an improved rotator cuff tear arthropathy model after 4 weeks, immunohistochemically stained in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

[0016] The model animal of shoulder arthropathy with rotator cuff tear according to the present embodiment is a rodent in which the supraspinatus muscle tendon, the infraspinatus muscle tendon, the superior joint capsule, and the long head tendon of the biceps brachii muscle in the glenohumeral joint on the same side are cut off. Examples of rodents include rats (Rattus norvegicus), mice (Mus musculus), and guinea pigs (Cavia porcellus). Since the anatomical similarity of the rotator cuff surroundings with humans is high, the rodent is preferably a rat. The following describes a model animal of shoulder arthropathy with rotator cuff tear using a rat as a rodent.

[0017] The age of the rat is not particularly limited, but is 10 weeks or more, or 11 weeks or more, preferably 12 weeks or more. The rat may be male or female. The location to be dissected in the model animal of rotator cuff tear shoulder arthropathy is shown in FIG. 1. The rotator cuff is composed of the supraspinatus, infraspinatus, teres minor, and subscapularis muscles. The supraspinatus originates from the supraspinatus fossa and the upper surface of the scapular spine of the scapula, and is inserted on the greater tuberosity of the humerus. The infraspinatus originates from the infraspinatus fossa and the lower surface of the scapular spine of the scapula, and is inserted on the greater tuberosity of the humerus. The upper joint capsule (also called the joint capsule ligament complex) originates from the neck and labrum of the scapula and its outer periphery to surround the joint cavity, and is attached to the greater tuberosity and lesser tuberosity. The long head tendon of the biceps brachii originates from the supraglenoid tubercle of the scapula, passes through the joint capsule and the intertubercular groove of the humerus, and is mostly attached to the radial tuberosity. In the model animal of rotator cuff tear shoulder arthropathy, the supraspinatus and infraspinatus tendons that compose the rotator cuff on the same side, the superior joint capsule, and the long head tendon of the biceps brachii are each severed.

[0018] Here, a method for producing a rotator cuff tear shoulder arthropathy model animal according to the present embodiment will be described. The method for producing a rotator cuff tear shoulder arthropathy model animal includes first to fourth steps. FIG. 2 shows an example of the method for producing a rotator cuff tear shoulder arthropathy model animal in the right glenohumeral joint of a rat. Preferably, the method for producing a rotator cuff tear shoulder arthropathy model animal is performed on anesthetized rats. Before the first step, the deltoid muscle is incised in a T shape as shown in FIG. 2A to expose the supraspinatus and infraspinatus muscle tendons.

[0019] In the first step, as shown in FIG. 2B and FIG. 2C, the muscular tendon of the supraspinatus muscle (hereinafter also simply referred to as "SSP") and the muscular tendon of the infraspinatus muscle (hereinafter also simply referred to as "ISP") are severed at the attachment portion of the humeral head, respectively. The severed portion can be performed by a known method using a scalpel, scissors, or the like. In the second step, the upper joint capsule (hereinafter also simply referred to as "capsule") including the superior glenohumeral ligament (hereinafter also simply referred to as "SGHL") at the glenohumeral joint, as shown by the dashed line in FIG. 2D, is severed. In the third step, the tendon of the long head of the biceps brachii (hereinafter also simply referred to as "LHB") at the glenohumeral joint is severed.

[0020] As shown in the following examples, the rotator cuff tear shoulder arthropathy model animal according to the present embodiment shows a significant change in OA about two weeks after surgery. Four weeks after surgery, the pathology of OA is more pronounced, and the subchondral bone is depressed. Fibrous pannus appears in the area covering the depressed subchondral bone. In addition, in the rotator cuff tear shoulder arthropathy model animal, the bone metabolism balance is inclined toward bone resorption, and a decrease in bone mass of the humeral head, which has been reported in CTA patients, is observed. Furthermore, in the rotator cuff tear shoulder arthropathy model animal, the expression of inflammatory cytokines and catabolic enzymes (cartilage matrix digestive enzymes) increases, similar to human OA.

[0021] The rotator cuff tear shoulder arthropathy model animal according to the present embodiment has a pathophysiology similar to that of human CTA, and can reproduce the pathology of rotator cuff tear shoulder arthropathy more faithfully. Although there are many unknowns about the natural course of human CTA, the rotator cuff tear shoulder arthropathy model animal allows for time-dependent observation of histological changes, particularly changes in the subchondral bone. In addition, the rotator cuff tear shoulder arthropathy model animal contributes to the elucidation of causative genes, causative proteins, disease aggravating factors, etc., as well as research and development of new therapeutic drugs, and research into the therapeutic effects and side effects of existing drugs. Furthermore, the rotator cuff tear shoulder arthropathy model animal allows for the development of regenerative medicine for bones, cartilage, ligaments, tendons, muscles, synovium, etc., treatment devices, surgical methods, surgical instruments, training methods, rehabilitation techniques, supports, taping methods, etc.

[0022] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0023] Male Sprague-Dawley (hereinafter also simply referred to as "SD") rats (manufactured by Japan SLC Co., Ltd.) were housed two per cage, given food and water ad libitum, and raised in a room with a controlled temperature of 23±1°C under a 12-hour light-dark cycle. In this example, 52 12-week-old rats (367±11 g) were used. All surgical procedures were performed under general anesthesia using 2% isoflurane mixed with oxygen, and penicillin (20000 U / kg) was administered subcutaneously to the rats to prevent infection. Buprenorphine (0.05 mg / kg, subcutaneous, every 12 hours) was administered to the rats after surgery for analgesia.

[0024] Humane endpoints were defined as difficulty eating and drinking, self-injury, abnormal posture, and significant impairment of quality of life due to lameness, but no animals were excluded because there were no adverse events, they ate well, and they did not lose weight.

[0025] A rotator cuff tenotomy (RCT) model was prepared as follows. The skin over the glenohumeral joint was incised on the right shoulder of SD rats (n=7 per time point), and the deltoid muscle was incised longitudinally at the humeral head to allow manipulation of the rotator cuff. The biceps muscle was left untreated. The SSP and ISP tendons were resected at their attachments at the humeral head. For sham surgery (hereafter simply referred to as "Sham"), the skin and deltoid muscle were incised on the left shoulder. The rats were sacrificed immediately after surgery (day 0), 1 week after surgery, and 4 weeks after surgery.

[0026] A modified rotator cuff tear arthropathy (hereinafter referred to as "mCTA") model was prepared as follows. The deltoid muscle was incised in a T-shape in the right shoulder of SD rats (n=7 for histological analysis at each time point, except for the 4th week, when an additional sample was added for macroscopic examination and bone histomorphometry, making n=17) (Fig. 2A), and the SSP muscle tendon and the ISP muscle tendon were cut at their attachments at the humeral head (Fig. 2B and Fig. 2C). The capsule containing the SGHL was then cut (dashed line in Fig. 2D), and the LHB was cut (Fig. 2D). The skin and deltoid muscle of the left shoulder were cut as a sham. The rats were sacrificed 1 week, 2 weeks, 4 weeks, and 8 weeks after surgery.

[0027] Glenohumeral joints and surrounding tissues were collected from sacrificed rats and fixed in 10% neutral buffered formalin at room temperature for 24 hours. They were then decalcified in K-CX AT solution (Pharma) at 4°C for 5 days and paraffin-embedded sections were prepared. For histological evaluation, the articular cartilage was divided into three regions: superior, middle, and inferior. The thickness of the articular cartilage was measured at 20 points in each region by Safranin O staining, and the average value was calculated. Alcian blue (pH 1.0) staining was performed to evaluate the changes in OA using the Murine Shoulder Arthritis Score (MSAS; see Non-Patent Document 2 above). Each measurement was performed twice by different practitioners.

[0028] For bone histomorphometry, calcein (8 mg / kg) was injected intraperitoneally into rats 5 and 1 days before sacrifice. Glycidyl methacrylate resin-embedded sections were stained with toluidine blue. The metaphysis of the humerus was analyzed using a Histometry RT Camera (System Supply Co., Ltd.) at Kureha Analytical Center. The subchondral bone of the humeral head was analyzed using a BZ-X710 / BZ-X700 Microscope System (Keyence Co., Ltd.).

[0029] For immunohistochemistry, sections were incubated with LABsolution (Polysciences) for antigen retrieval, endogenous peroxidase was inactivated with phosphate-buffered saline containing 3% hydrogen peroxide, and blocked with CAS-Block (Thermo Fisher Scientific). The primary antibodies included rabbit polyclonal anti-tumor necrosis factor (TNF)-α (1:200, bs-2081R, Bioss), rabbit polyclonal anti-interleukin (IL)-1β (1:200, bs-0812R, Bioss), rabbit polyclonal anti-IL-6 (1:200, bs-0782R, Bioss), rabbit polyclonal anti-matrix metalloproteinase (MMP)-3 (1:200, 17873-1-AP, Proteintech), rabbit polyclonal anti-MMP-13 (1:200, ab39012, Abcam), and rabbit polyclonal anti-ADAMTS (a disintegrin and metalloproteinase with thrombospondin The following antibodies were used: rabbit polyclonal anti-CD34 (1:200, bs-0646R, Bioss), mouse monoclonal anti-vimentin (1:1000, 60330-1-Ig, Proteintech), mouse monoclonal anti-leptin receptor (1:200, MA5-32685, Invitrogen), rabbit polyclonal anti-PDGFR α (1:200, bs-0231R, Bioss), mouse monoclonal anti-RUNX2 (1:200, D130-3, MBL International), and mouse monoclonal anti-RANKL (receptor activator of nuclear factor-kappa B ligand) (1:100, ab45039, Abcam).

[0030] Normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology) were used as negative controls for the primary antibodies. Histofine Simple Stain Rat MAX PO (MULTI) (414191, Nichirei Biosciences), a mixture of anti-mouse peroxidase-conjugated secondary antibodies and anti-rabbit peroxidase-conjugated secondary antibodies, was used to detect the signals of the primary antibodies. The signals were visualized using diaminobenzidine solution (415171, Nichirei Biosciences). Counterstaining was performed using Mayer's hematoxylin solution. Images of the sections were taken using a BZ-X710 / BZ-X700 microscope system. The number of signal-positive cells was divided by the total number of cells to quantify the positive staining rate. The average score of three independent areas was used for statistical analysis.

[0031] (result) First, we evaluated the degeneration of the humeral head articular cartilage in the RCT model. Histological evaluation by hematoxylin-eosin staining showed that the severed tendons of the SSP and ISP were reconnected by granulation tissue infiltrated with inflammatory cells one week after surgery, as shown in Figure 3A, and further repaired by scar tissue within four weeks. When the changes in OA in the shoulder of the RCT model were evaluated by MSAS, as shown in Figures 3B and 3C, the degree of staining with Alcian blue was slightly decreased and MSAS was slightly increased in the upper region of the humeral head cartilage, while the cartilage in the middle and lower regions was not affected (Wilcoxon rank sum test). This result indicates that the rat has the self-repairing ability of the rotator cuff tendons, preventing instability of the shoulder joint and direct collision between the humeral head and the acromion.

[0032] The humeral head of the mCTA model after 4 weeks is shown in Figure 4. In Figure 4, "SSC" indicates the subscapularis muscle. The glossy surface of the cartilage was generally lost, and it was faded and rough, especially in the upper region. When the characteristics of shoulder OA in the mCTA model were evaluated by Alcian blue staining, the detached rotator cuff tendon was repaired by scar tissue within 2 weeks, as shown in Figure 5. After 4 weeks, the subchondral bone collapsed, and the collapse then expanded. In the mCTA model, the roundness of the articular part of the humeral head, the cellularity of the chondrocytes, the morphology of the subchondral bone, and the stainability with Alcian blue were reduced, but from 4 weeks, fibrous pannus appeared and covered the damaged subchondral bone. The results of the degree of change in OA in the upper region in the mCTA model evaluated by MSAS are shown in Figure 6. In Figure 6, "S" indicates Sham and "C" indicates the mCTA model. In the evaluation of MSAS, Wilcoxon rank sum test was performed between the sham and mCTA models, and Kruskal-Wallis test and Steel-d'Owass test were performed between each group at different time points. As shown in Figure 6, the change in OA became significant in a time-dependent manner. Figure 7 shows images of sections stained with Safranin O from the sham and mCTA models after 4 weeks. In the mCTA model, abundant fibrous pannus was observed covering the collapsed subchondral bone.

[0033] The results of immunohistochemical staining for OA-related inflammatory cytokines and catabolic enzymes in the humeral head articular cartilage sections (superior region) of the mCTA model after 4 weeks are shown in Figure 8. In the mCTA model, the expression of TNF-α, IL-1β, IL-6, MMP-3, MMP-13 and ADAMTS-5 was significantly increased. Therefore, the pathophysiology of cartilage degeneration in the mCTA model was similar to OA.

[0034] Figure 9A shows a section of the humeral head stained with toluidine blue from a non-decalcified hard tissue section. The bone mass (BV (bone mass) / TV (total bone mass)), osteoclast number per unit area (Oc.N (osteoclast number) / BS (bone surface area)), mineralization rate (MAR), and bone formation rate (BFR / BS) in the subchondral bone of the mCTA model after 4 weeks are shown in Figures 9B, 9C, 9D, and 9E, respectively (Wilcoxon rank-sum test). The pathological condition of the humeral head in CTA patients, in which bone mass is reduced, was reproduced in the mCTA model after 4 weeks. In the mCTA model, bone resorption was enhanced and bone formation was unchanged, indicating that the bone metabolism balance was tilted toward bone resorption.

[0035] FIG. 10 shows the results of immunohistochemical staining of bone marrow-derived mesenchymal stem cell markers (CD34, vimentin, leptin receptor, and PDGFRα) and immature osteoblast markers (RUNX2 and RANKL) in pannus cells near the sunken subchondral bone in the humeral head of the mCTA model after 4 weeks. All bone marrow-derived mesenchymal stem cell markers were positive, suggesting that the origin of the pannus cells was mesenchymal stem cells. It was also shown that the pannus cells express RUNX2, a major regulator of osteoblast differentiation, and RANKL, which promotes osteoclast formation.

[0036] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for the purpose of explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not the embodiments. Various modifications made within the scope of the claims and the scope of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0037] INDUSTRIAL APPLICABILITY The present invention is useful for molecular pathological research of rotator cuff tear shoulder arthropathy, and for the development of therapeutic drugs, regenerative medicine, therapeutic devices, and the like.

Claims

1. A rodent in which the supraspinatus muscle tendon, the infraspinatus muscle tendon, the superior joint capsule, and the long head of the biceps brachii tendon at the same side of the glenohumeral joint are severed. Animal model of rotator cuff tear shoulder arthropathy.

2. The rodent comprises: It is a rat. The animal model of shoulder arthropathy caused by rotator cuff tear according to claim 1 .

3. The rat is 12 weeks old or older. The animal model of shoulder arthropathy caused by rotator cuff tear according to claim 2.

4. The subchondral bone is depressed or has been depressed The animal model of shoulder arthropathy caused by rotator cuff tear according to any one of claims 1 to 3.

5. The supraspinatus and infraspinatus tendons were dissected at the glenohumeral joint on the same side of the rodent. The upper joint capsule of the glenohumeral joint is dissected, The long head tendon of the biceps brachii is separated at the glenohumeral joint. A method for producing an animal model of shoulder arthropathy caused by rotator cuff tear.

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