KR101871673B1 - Composition comprising nucleic acid and chitosan for repair of rotator cuff tear - Google Patents

Abstract

The present invention relates to a composition containing a nucleic acid and a chitosan for rotator cuff tear recovery, and more particularly, a composition for rotator cuff tear recovery prepared by mixing nucleic acid and chitosan has a sol-gel transition according to temperature , It was confirmed that the tissue restoration effect was higher than that of the conventional single composition, and the tissue restoration effect was sustained by slowing the decomposition rate of the composition and increasing the residence time of the composition at the rupture site.
Thus, it is expected that a therapeutic composition having a remarkably high restorative effect on ruptured cancellous tissue can be developed by using the composition of the present invention comprising the nucleic acid and the chitosan.

Description

Technical Field [0001] The present invention relates to a composition for the treatment of rotator cuff tear comprising nucleic acid and chitosan,

The present invention relates to a composition for rotator cuff tear recovery comprising a nucleic acid and chitosan.

There are four muscles in the shoulder that are necessary to move the shoulders (extremity muscles, extreme lower muscles, scapular muscles, and wishes muscles), which is called a rotator cuff dog. The rotator cuff converts the vertical shear force of the strong deltoid to a smooth abduction shear force and serves as a protective barrier to prevent the humeral head from contacting the lower portion of the acromion (Kang J.D., et al., 2007).

The rotator cuff injury is sometimes caused by acute trauma, but most of it is known to progress slowly without any special trauma. In other words, the rotator cuff disease is basically a degenerative disease caused by aging phenomenon. However, external factors include instability of the shoulder joint, arthritis of the acromioclavicular joint, osacromiale, collateral ligament collision, This can be caused by this. In addition, rotator cuff damage is not only caused by external compression due to collision (impingement syndrome) in the lower part of the acromion, but also by various factors such as intrinsic degeneration of the rotator cuff itself or influences of the inflammatory reaction It is known. These pathological changes can lead to rupture of the rotator cuffs, resulting in serious disruption of daily activities and work activities, if the arm is overused or accompanied by mild trauma (Cho N.S., et al., 2012).

Symptoms of rotator cuff injury include pain, muscle atrophy, limitation of joint motion, weakness in shoulder muscle strength, and tenderness in the anterolateral portion of the shoulder. If the degree of rupture is severe, the ruptured part is externally promoted. Also, if you try to raise your sore arm with your right arm, you will climb up, but you will not be able to keep your arms weakened due to the rupture, and your sore arm will fall off or you will have a shoulder pain.

There is a difference in the clinical characteristics and treatment of the rotator cuff injuries according to each stage from the first stage to the third stage of the shoulder. In stage 1, edema and hemorrhage of the rotator cuff are present. There are many cases in the age group below 25 years, and conservative treatment is possible. The second stage is the stage of fibrosis and tendinitis of the rotator cuff. It is common in the age group from 25 to 40 years. Conservative treatment may improve, but severe symptoms require surgery. The third stage is the stage of degenerative spondylolisthesis and rupture of the rotator cuff, which is more common in patients over 40 years of age. The symptoms continue to worsen and require surgery.

The treatment of rotator cuff tear is conservative and operative treatment. In Korea, many surgical procedures are performed. Endoscopic knee repair and open repair are the surgical methods. Regardless of the technique, there is always the possibility of postoperative complications such as failure of operation and infection, and surgical scar remains in the deltoid muscle. In addition, several months of rehabilitation training is required after surgery, and the pain may not be completely eliminated by surgery, or movement may not be fully restored due to rigidity of the joints (Lee J. H., et al., 2011).

Conservative treatment methods include relaxation, pain relief, use of topical steroid injections, and physical therapy as methods for relieving inflammation and pain. Stretching exercises and restoration of function to restore softness of soft tissues (Jung HJ, et al., 2012), which is the most common muscle strength training program in Korea. In the case of drug therapy, nonsteroidal anti-inflammatory analgesics have been used orally or topically, or steroidal use has been used. However, these drug treatments have a short-term effect but a high recurrence rate and side effects. In addition, non-surgical treatment methods such as physical therapy methods such as ultrasonic thermal therapy and electrotherapy are restricted to symptom alleviation such as pain reduction, and there is a limit in not being a therapeutic method for positively solving the cause.

Recently, a new treatment method based on tissue regeneration effects such as platelet-rich plasma injection therapy has been introduced as a non-surgical treatment method, and it has been reported that a therapeutic effect is actually obtained in rotator cuff tear .

Accordingly, the present inventors have completed the present invention by confirming that a composition containing nucleic acid and chitosan in the course of studying a composition for rotator cuff tear repair has excellent pain relieving effect by rotator cuff tearing and excellent restorative effect of torn rotator cuffs there was.

As a prior art, Korean Patent Registration No. 2015-0131864 discloses a pharmaceutical composition for preventing or treating rotator cuff tearing which contains a diene fragment mixture, Korean Patent No. 1536134 discloses a pharmaceutical composition for preventing or treating soft tissue swelling However, there is no description about a composition for tearing the rotator cuff containing a nucleic acid and chitosan, which is different from the present invention in its constitution.

Korean Patent Laid-Open Publication No. 2015-0131864, a pharmaceutical composition for preventing or treating rotator cuff tearing, comprising a mixture of dienes fragments separated from semen or testicles of fish, Korean Patent No. 1536134, Manufacturing Method of Matrix for Soft Tissue Recovery, Registered on Jul. 07,

Cho N.S., et al., Impingement Syndrome and Rotator Cuff Tear: Etiology, Journal of Korean Arthroscopy Society, 16 (1), 72-78, 2012. Jung H.J., et al., Conservative Treatment of Impingement Syndrome and Rotator Cuff Tear, Journal of Korean Arthroscopy Society, 16 (1), 79-86, 2012. Kang J. D., et al., Rotator Cuff Tears Syndrome, Korean J. Orthop. Manu. Ther., 12 (1), 67-72, 2007. Lee J. H., et al., Case Report of Acute Traumatic Rotator Cuff Tear Treatment in Traditional Korean Medicine, Journal of Pharmacopuncture, 14 (4), 53-58, 2011.

It is an object of the present invention to provide a composition for rotator cuff tear recovery containing a nucleic acid and chitosan.

It is still another object of the present invention to provide a method for preparing a composition for tearing a rotator cuff containing a nucleic acid and chitosan.

The present invention relates to a composition for rotator cuff tear recovery comprising a nucleic acid and chitosan.

The nucleic acid may be present in an amount ranging from 0.1% to 3% by weight based on the total weight of the composition.

The content of the chitosan may be 0.001 wt% to 0.1 wt% based on the total weight of the composition.

The nucleic acid and chitosan may be in a weight ratio of 1: 1 to 3000: 1.

The nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture thereof. Preferably, it is deoxyribonucleic acid (DNA).

The deoxyribonucleic acid may be selected from oligonucleotides, polynucleotides and polydeoxyribonucleotides.

The nucleic acid may have a molecular weight of 1 kDa to 100,000 kDa. Preferably from 10 kDa to 10,000 kDa, and more preferably from 50 kDa to 3,500 kDa.

The chitosan may have a molecular weight of 3 kDa to 1,000 kDa.

The composition may contain a water-soluble polymer as an additional component.

The water-soluble polymer may be selected from the group consisting of hyaluronic acid, chondroitin sulfate, glycogen, dextrin, dextran, dextran sulfate, hydroxypropyl methylcellulose Alginic acid, chitin, pullulan, collagen, gelatin and hydrolyzates thereof, polyvinyl alcohol, polyvinyl pyrrolidone (polyvinyl pyrrolidone) ), Polyacrylic acid, and a carboxylvinyl polymer. It is preferably hyaluronic acid.

The composition can be injected through a syringe.

The composition may be a hydrogel in which the sol-gel transition occurs depending on the temperature.

Hereinafter, the present invention will be described in detail.

The composition for rotator cuff tearing containing the nucleic acid and chitosan comprises the steps of: i) preparing a nucleic acid storage solution by dissolving the nucleic acid in a buffer and stirring at 40 ° C to 70 ° C for 30 minutes to 1 hour; ii) dissolving chitosan in an acidic buffer solution to prepare a chitosan storage solution; iii) The nucleic acid storage solution of step i) and the chitosan storage solution of step ii) are mixed at a weight ratio of nucleic acid and chitosan of 1: 1 to 3000: 1, and the mixture is incubated at 40 to 70 ° C for 30 minutes to 1 hour Stirring; And iv) lowering the nucleic acid-chitosan mixture of step iii) to room temperature with stirring; . ≪ / RTI >

The buffer solution used for preparing the nucleic acid storage solution may include sodium phosphate dibasic dodecahydrate, sodium chloride, magnesium chloride, potassium chloride, phosphate buffered saline phosphate buffer saline or HEPES (N- (2-hydroxyethyl) -piperazine-N'-2-ethanesulfonic acid) buffer solution, preferably sodium phosphate dibasic dodecahydrate, , Sodium phosphate dibasic dodecahydrate of 5 to 300 mM may be used, but is not limited thereto.

The acidic buffer solution used for preparing the chitosan storage solution includes acetic acid, hydrochloric acid, ascorbic acid, lactic acid, nitric acid, glutamic acid, Or formic acid, preferably acetic acid, and most preferably 10 to 300 mM of acetic acid may be used, but is not limited thereto.

The composition for rotator cuff tearing containing the nucleic acid and chitosan may be a thermosensitive hydrogel in which sol-gel transition occurs depending on temperature.

In the thermosensitive hydrogel, a phenomenon in which the sol changes to a gel or a gel changes to a gel is called a gelation, and the gelation in the present invention Has a viscoelasticity and is defined as a polymer having a three-dimensional net structure as the temperature increases and remaining in a state of being insoluble in a solvent.

The composition for the tearing of the rotator cuff having the thermosensitive hydrogel property is injected into the rupture site, and the viscoelasticity is recovered by the body temperature to gel, thereby increasing the residence time of the composition at the rupture site, thereby improving the tissue restoration effect. Also, by slowing the decomposition rate of the composition due to the gelation of the composition, a long-term therapeutic effect can be obtained.

The composition for the tearing of rotator cuffs containing the nucleic acid and the chitosan has a high stability that does not cause layer separation while maintaining homogeneous state. The nucleic acid and the chitosan have a weight ratio of 1: 1 to 3000: 1 . Preferably from 10: 1 to 300: 1. More preferably 10: 1 to 100: 1.

The nucleic acid may be present in an amount ranging from 0.1% to 3% by weight based on the total weight of the composition. Preferably 1% by weight to 2% by weight.

The nucleic acid may have a molecular weight of 10 kDa to 100,000 kDa, preferably 10 kDa to 10,000 kDa, and most preferably 50 kD to 3,500 kDa. When the molecular weight of the nucleic acid is less than 10 kDa, it is difficult to control the decomposition rate of the gel. When the molecular weight exceeds 100,000 kDa, the viscosity of the gel is difficult to control.

The nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture thereof. It is preferably a deoxyribonucleic acid.

The deoxyribonucleic acid may be an oligonucleotide, a polynucleotide, or a polydeoxyribonucleotide.

In addition, the content of the chitosan may be 0.001 wt% to 0.1 wt%, preferably 0.01 wt% to 0.1 wt%, based on the total weight of the composition.

The chitosan preferably has a molecular weight of 3 kDa to 1,000 kDa, but is not limited thereto.

The mixture of the nucleic acid and the chitosan is mixed such that the weight ratio of the nucleic acid and the chitosan is 1: 1 to 3000: 1, wherein the content of the nucleic acid is 0.1 wt% to 3 wt% based on the total weight of the composition, Is in the range of 0.001 wt% to 0.1 wt% based on the total weight of the composition.

The composition containing the nucleic acid and the chitosan may contain a water-soluble polymer as an additional component.

The water-soluble polymer may be added as a composition for treating tears of a rotator cuff containing a nucleic acid and chitosan in order to control the degradation rate and to enhance the therapeutic effect

The water-soluble polymer is soluble or decomposable in vivo and may be selected from the group consisting of hyaluronic acid, chondroitin sulfate, glycogen, dextrin, dextran, dextran sulfate, Proteins such as polysaccharides such as hydroxypropyl methylcellulose, alginic acid, chitin and pullulan, collagen, gelatin and hydrolyzates thereof, poly Synthetic polymer compounds such as polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid and carboxylvinylpolymer can be used, and hyaluronic acid is preferable.

The composition containing the nucleic acid containing the water-soluble polymer as an additional component and the chitosan and the composition containing the nucleic acid, the chitosan and the water-soluble polymer may be mixed in a weight ratio of 1: 3000: 1: 1 to 3,000. Preferably 10 to 1,000: 1: 10 to 1,000, and more preferably 25 to 100: 1: 25 to 100.

The hyaluronic acid includes a linear or crosslinked form and may have a molecular weight of 20 kDa to 10,000 kDa, preferably 500 kDa to 3,000 kDa, but is not limited thereto.

The compositions for the treatment of tears of rotator cuffs containing the nucleic acid and chitosan can inhibit fat degeneration and inflammatory reaction due to rotator cuff tear and improve the continuity and parallelability of the rotator cuff tissues to improve the repair effect of rotator cuff tissues. .

The present invention relates to a composition containing a nucleic acid and a chitosan for rotator cuff tear recovery, and more particularly, a composition for rotator cuff tear recovery prepared by mixing nucleic acid and chitosan has a sol-gel transition according to temperature , It was confirmed that the tissue restoration effect was higher than that of the conventional single composition, and the tissue restoration effect was sustained by slowing the decomposition rate of the composition.

Thus, the composition for the treatment of tears of the rotator cuff of the present invention can be directly injected into the ruptured region requiring treatment, slows the decomposition rate of the composition through the gelation at the injected site, and increases the residence time of the drug to the ruptured region The tissue repair effect can be sufficiently exhibited, and it is expected that an excellent therapeutic effect can be obtained.

Fig. 1 shows the results of checking the physical properties of the composition for repairing rotatory tear openers according to the present invention. Each of the composition for rotator cuff tearing was prepared and allowed to stand for 3 days. Then, transparency of the composition, formation of precipitate and separation of layers were confirmed.
FIG. 2 shows the results of confirming the formation of a hydrogel in which the sol-gel transition occurs according to the temperature of the composition for repairing rotatory tear gas according to Example 1-1 of the present invention.
FIG. 3 shows a method of measuring a maximum tensile strength when a treated rotator cuff is ruptured according to the composition for repairing a rotator cuff tear of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Preparation of composition for rotator cuff tear recovery containing nucleic acid and chitosan >

In the case of nucleic acid stock solution, the nucleic acid was prepared by dissolving in 190 mM sodium phosphate dibasic dodecahydrate buffer solution for 30 minutes or more using a heat agitator at 70 ° C.

The chitosan stock solution was prepared using 100 mM acetic acid.

The prepared nucleic acid and chitosan storage solution were mixed and stirred in a heat agitator at 70 ° C for 30 minutes. Thereafter, the temperature was lowered to room temperature and the mixture was stirred for 1 hour to prepare a composition for rotator cuff tear recovery containing nucleic acid and chitosan. At this time, the concentrations of nucleic acid and chitosan were adjusted to the concentrations shown in Table 1 below.

Configuration Final concentration (% by weight) Mixing ratio (weight ratio) Nucleic acid Chitosan Nucleic acid Chitosan Example 1-1 1.5 0.015 100 One Examples 1-2 0.1 0.001 100 One Example 1-3 0.1 0.1 One One Examples 1-4 3 0.001 3000 One Examples 1-5 3 0.1 30 One

< Example  2. Comprising nucleic acids, chitosan and hyaluronic acid Rotator  Preparation of composition for rupturable recovery>

In the case of the nucleic acid storage solution, the nucleic acid was prepared by dissolving the nucleic acid in a buffer solution of 190 mM sodium phosphate dibasic dodecahydrate at 70 ° C for 30 minutes or more.

The chitosan stock solution was prepared using 100 mM acetic acid.

The prepared nucleic acid and chitosan storage solution were mixed and stirred in a heat agitator at 70 ° C for 30 minutes. The hyaluronic acid raw material was further added to the mixed solution of nucleic acid and chitosan, stirred for 1 hour at 60 ° C in a heat agitator, and then stirred at room temperature for 3 hours to prepare a composition for rotator cuff tear recovery containing nucleic acid, chitosan and hyaluronic acid. At this time, the concentrations of nucleic acid, chitosan and hyaluronic acid were adjusted to the concentrations shown in Table 2 below.

Configuration Final concentration (% by weight) Mixing ratio (weight ratio) Nucleic acid Chitosan Hyaluronic acid Nucleic acid Chitosan Hyaluronic acid Example 2-1 1.5 0.03 1.5 50 One 50 Example 2-2 0.1 0.001 0.1 100 One 100 Example 2-3 0.1 0.001 3 100 One 3000 Examples 2-4 0.1 0.1 0.1 One One One Example 2-5 0.1 0.1 3 One One 30 Examples 2-6 3 0.001 0.1 3000 One 100 Examples 2-7 3 0.001 3 3000 One 3000 Examples 2-8 3 0.1 0.1 30 One One Examples 2-9 3 0.1 3 30 One 30

The hyaluronic acid having a molecular weight of 1,000 kDa is used, but it is also possible to use a hyaluronic acid having a molecular weight of 20 kDa to 10,000 kDa.

&Lt; Comparative Example 1 > Preparation of composition for comparison of rotator cuff tear water for comparison>

The compositions for comparison of the rotator cuff tears according to the contents of the components and their contents and mixing ratios in Table 3 below were prepared. The same method as in Example 1 and Example 2 was used for the manufacturing method.

Configuration Final concentration (% by weight) Mixing ratio (weight ratio) Nucleic acid Chitosan Hyaluronic acid Nucleic acid Chitosan Hyaluronic acid Comparative Example 1-1 2 0 0 One 0 0 Comparative Example 1-2 0 One 0 0 One 0 Comparative Example 1-3 1.5 0 1.5 One 0 One Comparative Example 1-4 1.5 0.0001 0 15000 One 0 Comparative Example 1-5 1.5 One 0 1.5 One 0 Comparative Example 1-6 0.001 0.0001 0.001 10 One 10 Comparative Example 1-7 0.001 0.000001 0.001 1000 One 1000 Comparative Example 1-8 1.5 0.5 1.5 3 One 3 Comparative Example 1-9 1.5 0.00075 1.5 2000 One 2000 Comparative Example 1-10 6 0.03 1.5 200 One 50 Comparative Example 1-11 6 0.03 6 200 One 200 Comparative Example 1-12 0 0.03 1.5 0 One 50 Comparative Example 1-13 0 0.03 6 0 One 200

< Experimental Example  One. Rotator  Confirming the physical properties of the composition for rupturing water>

(Examples 1-1 to 1-5), Example 2 (Examples 2-1 to 2-9), and Comparative Example 1 (Comparative Examples 1-1 to 1-13) ) Was used to confirm gelation, stability of the gel and solubility of the gel.

Each composition was mixed and allowed to stand for 3 days. Then, the transparency and gelation state of the composition were visually observed. The gelation degree was maintained at 1 DEG C from 24 DEG C to 40 DEG C for 1 minute using a rheometer The stability of the gel was confirmed by the formation of precipitates and the separation of layers. Whether or not the gel was soluble was evaluated by gelling the composition for rotary tear rupture recovery of Example 1, Example 2, and Comparative Example 1 in an aqueous solution at 37.5 ° C and stirring the mixture at 400 rpm for 5 minutes while maintaining the temperature at 37.5 ° C. , And the results are shown in Table 4 and Fig.

Configuration Results after 3 days At 37.5 [deg.] C for 5 minutes
Stirring result Viscoelastic Produce sediment Layer separation Dissolution of gel Example 1-1 ○ × × × Examples 1-2 ○ × × × Example 1-3 ○ × × × Examples 1-4 ○ × × × Examples 1-5 ○ × × × Example 2-1 ○ × × × Example 2-2 ○ × × × Example 2-3 ○ × × × Examples 2-4 ○ × × × Example 2-5 ○ × × × Examples 2-6 ○ × × × Examples 2-7 ○ × × × Examples 2-8 ○ × × × Examples 2-9 ○ × × × Comparative Example 1-1 ○ × × ○ Comparative Example 1-2 × × × ○ Comparative Example 1-3 ○ × × ○ Comparative Example 1-4 ○ × × ○ Comparative Example 1-5 × ○ × ○ Comparative Example 1-6 × × × ○ Comparative Example 1-7 × × × ○ Comparative Example 1-8 ○ ○ × ○ Comparative Example 1-9 ○ × × ○ Comparative Example 1-10 × ○ × × Comparative Example 1-11 × ○ × × Comparative Example 1-12 ○ ○ ○ ○ Comparative Example 1-13 ○ ○ ○ ○

Referring to Table 4 and FIG. 1, it can be seen that, in the case of the composition for repairing tear openers of Examples 1 and 2, the viscoelasticity is maintained even when three days have elapsed without occurrence of precipitate formation and delamination In the case of the solubility of the gel, a gel was formed at 37.5 DEG C, and the formed gel was observed to be maintained continuously.

However, in the case of the composition for comparison of the rotator cuff tear of Comparative Example 1, it was impossible to measure the viscoelasticity due to precipitation, layer separation, or partial lump formation, and gel solubility at 37.5 ° C It was confirmed that they all dissolved within 5 minutes even if gelation occurred. Particularly, in the case where the nucleic acid is not contained in the composition for comparison with Comparative Example 1-12 and Comparative Example 1-13, the composition has viscoelasticity, but precipitate formation and layer separation phenomenon occur. Was completely dissolved within 5 minutes after the formation of the catalyst, and the stability was poor (see Table 4).

As a result, it was found that the composition for the treatment of rotator cuffs according to the present invention exhibited temperature sensitivity according to temperature changes and kept the shape of the gel even after gelation, It can be expected that the treatment effect of the rotator cuff tear can be improved by increasing the therapeutic effect of the injured rotator cuff region by increasing the drug decomposition rate and slowing the decomposition rate of the composition.

Experimental Example 2. Determination of sol-gel transition according to temperature &gt;

Hydrogel formation in which the sol-gel transition occurred in accordance with the temperature change of the composition for repairing tear openers of Example 1, Example 2, and Comparative Example 1 was confirmed.

A rheometer was used to confirm the sol-gel transition. The measurement conditions were as follows: G '(elastic) and G' (length) were maintained for 1 minute while increasing the temperature by 1 ° C from 24 ° C to 40 ° C with a PU 20, a gap of 0.5 mm, a 0.1 Hz and a 1% stress strain, (Viscosity) of each composition was measured. The change in the sol-gel phase before and after 36 ° C was visually observed by adding a temperature to each composition and is shown in Fig.

As shown in FIG. 2, it was observed that the composition for rotator cuff rupture repair of Example 1-1 was in the form of a sol at a temperature lower than 36 ° C, whereas it was gelled at a temperature higher than 36 ° C. These results were confirmed to be the same in both of the compositions for repairing rotatory can openers of Examples 1 and 2. [

Accordingly, the composition of the present invention has temperature-sensitive properties in which a sol-gel transition occurs depending on the temperature, so that the composition is gelled by the body temperature after injected into the rotator cuff tear region and the retention time of the drug is increased It can be expected that the restoration effect of the tissue can be maximized.

<Experimental Example 3> Preparation of an animal model of rotator cuff tear:

Male Sprague-Dawley rats were randomly assigned to each experimental group. The rats in the experimental group except the normal group were exposed to the right shoulder and the infraspinatus, one of the rotator cuffs, was cut and wrapped with a penrose drain. The skin was sutured and maintained for 4 weeks to induce chronic rotator cuff tear rotator cuff tear model.

<Experimental Example 4> Injection and biopsy of composition for tearing of rotator cuff canal>

After rupturing the ruptured rotator cuffs by opening the right shoulder of the animal model of chronic rotator cuff produced in Experimental Example 3, 0.5 ml of the composition for the tear repair of the present invention was injected, and after 3 or 6 weeks Lt; / RTI &gt; After 3 or 6 weeks, histological examination was performed on the rotator cuff of each animal model. Histopathologically, tissues collected in 10% neutral buffered formalin were immobilized for more than 24 hours, and debris and paraffin invasion were performed to identify the proximal 0.3 cm region of the musculotendinous junction.

<Experimental Example 5> Histopathological observation>

Experimental Example 5-1. Identification of biocompatibility and degree of healing of composition for rotator cuff tear repair by fatty degeneration and histological examination of inflammatory reaction

If the rotator cuff tears are damaged, internal metamorphosis and inflammation of the rotator cuff tend to occur. If the sphincter of the rotator cuff tears fails, fat degeneration and inflammatory reaction of the rotator cuff tend to occur, . Thus, after treatment of the composition for repairing rotator cuff tear of the present invention, changes in fat degeneration and inflammatory reaction were observed to confirm the degree of repair of rotator cuff tears.

In order to confirm the fat degeneration of the tissue section, the number of adipocytes was changed to S100, the number of macrophages was changed to CD68 to identify the inflammatory reaction, and the number of stained cells was statistically processed.

division Fat cells (S100) Macrophage (CD68) 3 weeks 6 weeks 3 weeks 6 weeks Example 1-1 67.1 + - 14.0 44.4 + - 21.0 14.2 ± 21 10.7 ± 17 Example 2-1 53.7 ± 24.0 36.7 ± 28 11.6 ± 18 8.5 ± 12 Comparative Example 1-1 102.1 ± 27.0 92.6 ± 33 18.6 ± 19 17.5 ± 18 Comparative Example 1-2 139.4 ± 36.0 176.7 ± 42.0 22.1 ± 15.0 21.4 ± 21.0 Comparative Example 1-3 94.3 ± 21.0 81.1 ± 25 17.4 ± 17 16.7 ± 20 Untreated group 150.3 + - 54.0 193.7 ± 51.0 24.5 ± 17.0 23.6 ± 14.0 Normal group 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

As shown in Table 5, the number of adipocytes and macrophages increases with time in the animal model of rotator cuff tear (untreated group), indicating that the lesion is deteriorating. On the other hand, the number of adipocytes and macrophages decreased with the lapse of time in the group treated with the compositions for treating rotator cuffs of Example 1-1, Example 2-1, Comparative Example 1-1 and Comparative Example 1-3 However, in the group treated with the composition for treating rotator cuff tears of Comparative Example 1-2, the number of adipocytes and macrophages was increased similarly to the untreated group, and there was no tissue repair effect of the lesion. Further, in the case of the group treated with the compositions for the treatment of the rotator cuff tear in Examples 1-1 and 2-1, the group treated with the composition for tear rupture repair of Comparative Examples 1-1 and 1-3 The number of adipocytes and macrophages decreased more.

Thus, the composition for rotator cuff repair of the present invention is excellent in biocompatibility, and the drug retention property is increased at the tear site of the rotator cuff, so that the tissue restoration effect is remarkably improved.

Experimental Example  5-2. Rupture through histological examination of collagen fibers Rotator  Check the degree of healing

The degree of healing of the ruptured rotator cuffs was confirmed by histological examination of the continuity and regular parallelism of the collagen fibers of the bone - tendon joints.

Masson's trichrome staining and H & E (hematoxyline and eosin) staining were used to confirm the degree of collagen fiber continuity and regular parallelism of tissue sections. (0: 25%, 1: 25-50%, 2: 50-75%, 3:> 75%) depending on the degree of collagen fiber continuity and regular parallelism of the stained tissue sections. The number of rats in the rats was checked to evaluate the degree of healing of ruptured rotator cages. The results are shown in Table 6.

division Continuation of Collagen Fibers at Bone-tendon Boundary The parallelism of the collagen fibers at the bone-tendon boundary 3 weeks 6 weeks 3 weeks 6 weeks 0
step One
step 2
step 3
step 0
step One
step 2
step 3
step 0
step One
step 2
step 3
step 0
step One
step 2
step 3
step Example 1-1 0 2 2 0 0 0 2 2 0 One One 2 0 0 2 2 Example 2-1 0 One 2 One 0 0 One 3 0 0 2 2 0 0 One 3 Comparative Example 1-1 0 3 One 0 0 2 2 0 0 3 One 0 0 2 2 0 Comparative Example 1-3 0 2 2 0 0 One 3 0 0 One 2 One 0 0 3 One radish
Treated group 2 2 0 0 One 3 0 0 One 3 0 0 0 4 0 0

As shown in Table 6, in the animal model (untreated group) with rotator cuff tear, the degree of continuity and parallelism of the collagen fibers at the bone-tendon boundary was found to be in the 0 and 1 stages until 6 weeks. However, the compositions of Examples 1-1 and 2-1 and Comparative Examples 1-1 and 1-3 showed that the degree of continuity and parallelism of the collagen fibers at the bone-tendon boundary was 6 It was in stages 1, 2, and 3 until the week. In particular, it was found that the degree of change in continuity and parallelism of collagen fibers was higher in Examples 1-1 and 2-1 than in Comparative Examples 1-1 and 1-3.

Accordingly, it was confirmed that the composition for repairing rotator cuff tear of the present invention increases the drug retention at the tear site of the rotator cuff to improve the continuity and parallelism of the collagen fiber, and thus the tissue restoration effect is exhibited.

&Lt; Experimental Example 6: Measurement of tensile strength of rotary rods &

The tensile strength at the time when the treated rotator cuffs ruptured according to the compositions of Examples 1 and 2 of the present invention were confirmed by using the Instron 3343 instrument shown in Fig.

The rat rotator cuff was fixed with a pneumatic grip and tensile load was applied to measure the maximum tension when ruptured. At this time, the pattern of rupture appears in two forms: an insertional tear at the junction of the bone and the tendon, and a midsubstance tear at the middle of the tendon. The type of rupture is distinguished by the degree of strength of the joint. Midsubstance tear occurs when there is a strong bond in the bone-tendon, and Insertional Tear occurs when there is a relatively weak bond in the bone-tendon. It is known that when the midsubstance tear occurs, the tissue is strengthened.

Before the tensile test, a total load of 1N was applied and pretreated by applying a cyclic load of 10 cycles at a rate of 0.1 mm / s at 5% strain. After pretreatment, the stress-relaxation response was tested at a slope of 5% at a strain rate of 2.5% / s, after a 300 second wait and then returned to a full load of 1 N, followed by a quasi-static failure load A quasi-static load-to-failure test was performed at 0.1 mm / s. Load and displacement were recorded at 100 Hz.

Power analysis was carried out by setting the minimum number of samples required to detect a difference of 20% in the ratio of the peak failure load to 4 and using power of 0.8 and 0.05 (SD) estimated as an alpha value of 10%. The nonparametric analysis was used to compare the differences in the parameters between the two groups. The Mann-Whitney test was used for independent samples and the SPSS version 13.0 (SPSS, Chicago, IL) Program. The results are shown in Table 7.

division Breaking load Rupture pattern (Insertion rupture: Medium rupture) right left side right left side Example 1-1 20.24 + - 3.72 23.34 + - 4.85 2: 5 0: 7 Example 1-3 19.47 + - 4.21 23.64 + - 4.41 3: 4 0: 7 Examples 1-4 19.71. + -. 3.87 23.38 + - 4.29 2: 5 0: 7 Example 2-1 21.12 ± 3.62 22.94 + - 6.51 1: 6 0: 7 Examples 2-7 20.94 + - 4.34 23.34 + - 5.34 2: 5 0: 7 Comparative Example 1-1 14.84 + - 6.55 23.02 + - 6.04 5: 2 0: 7 Comparative Example 1-3 15.42 + - 6.57 23.14 + - 4.61 4: 3 0: 7 Untreated group 9.59 + - 6.44 23.58 + - 4.36 6: 1 0: 7 Normal group 24.06 ± 5.97 23.11 + 4.12 0: 7 0: 7 Right: Animal model of rotator cuff tear (right shoulder)
Left: normal arm animal model (left shoulder)

In Table 7, in the case of the group treated with the composition for repairing rotator cuff tear of Example 1-1, Example 1-3, Example 1-4, Example 2-1, and Example 2-7, The fracture load and tear pattern of the right shoulder of the right shoulder showed a similar measured value to the fracture load and tear pattern of the normal shoulder after 6 weeks. It was found that the value was significantly increased compared to the group treated with the composition for dog rupture.

In addition, the rotatory canopy rupture pattern in the group treated with the compositions for the treatment of rotator cuff tear in Examples 1-1, 1-3, 1-4, 2-1, and 2-7 was weak It was found that the ratio of the intermediate tear pattern showing stronger bond than the insert tear pattern indicating the bond was high, and the tensile strength of the rotator crown was strengthened and the rotator cuff tear was restored.

As a result, when the composition of the present invention was treated, it was found that the tensile strength of the rotator cages was increased with the lapse of time, and the excellent repair of the rotator cuff tear was achieved by restoration of the ruptured rotator cage tissues there was.

Claims (16)

From 0.1% to 3% by weight of nucleic acid and from 0.001% to 0.1% by weight of chitosan based on the total weight of the composition,
Wherein the weight ratio of the nucleic acid and the chitosan is 1: 1 to 3000: 1. delete delete delete The method according to claim 1,
Wherein the nucleic acid is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture thereof. 6. The method of claim 5,
Wherein the nucleic acid is deoxyribonucleic acid (DNA). The method according to claim 6,
Wherein the deoxyribonucleic acid is selected from oligonucleotides, polynucleotides, and polydeoxyribonucleotides. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein the nucleic acid has a molecular weight of 1 kDa to 100,000 kDa. 9. The method of claim 8,
Wherein the nucleic acid has a molecular weight of 10 kDa to 10,000 kDa. 10. The method of claim 9,
Wherein the nucleic acid has a molecular weight of 50 kDa to 3,500 kDa. The method according to claim 1,
Wherein the chitosan has a molecular weight of 3 kDa to 1,000 kDa. The method according to claim 1,
Wherein the composition contains a water-soluble polymer as an additional component. 13. The method of claim 12,
The water-soluble polymer may be selected from the group consisting of hyaluronic acid, chondroitin sulfate, glycogen, dextrin, dextran, dextran sulfate, hydroxypropyl methylcellulose Alginic acid, chitin, pullulan, collagen, gelatin and hydrolyzates thereof, polyvinyl alcohol, polyvinyl pyrrolidone (polyvinyl pyrrolidone) Wherein the composition is at least one selected from the group consisting of polyacrylic acid, carboxyvinylpolymer, and the like. 14. The method of claim 13,
Wherein the water-soluble polymer is hyaluronic acid. The method according to claim 1,
Wherein the composition is injected through a syringe. The method according to claim 1,
Wherein the composition is a hydrogel in which a sol-gel transition occurs depending on the temperature.

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