Application of plasmid composition in preparation of medicine for promoting healing of dental wound area

Abstract

The invention belongs to the field of biological agents, and particularly relates to application of a plasmid composition in preparation of a medicine for promoting healing of a dental wound area. The plasmid group composition is a DNA (deoxyribonucleic acid) plasmid composition containing SEQ ID NO: 1 or a DNA plasmid composition containing more than 90% of similarity with the sequence of SEQ ID NO: 1. The plasmid composition provided by the invention can shorten the repair and reconstruction time of alveolar bone and maintain better bone density, alveolar ridge height and width; the plasmid composition is free of obvious local stimulation, high in safety and good in application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological therapy, and particularly relates to application of a plasmid composition in preparation of a drug for promoting healing of a tooth wound area. BACKGROUND

[0002] Although people's awareness of oral health protection is increasing, teeth may still be missing due to reasons such as caries, periodontal disease, trauma, etc. In the past, when teeth were missing, removable denture or fixed bridge repair was the preferred method. With the rapid development of oral implantology, more and more patients begin to choose dental implant repair technology as one of the preferred solutions. Dental implant repair technology has become an advanced repair method commonly used in clinical practice, which can be used for the repair of single, multiple or full tooth loss, effectively solving different forms of missing problems. Implant denture can stably maintain the integrity of the alveolar bone, and at the same time, it will not damage the adjacent teeth; it has excellent chewing effect, greatly improving the patient's food intake capacity; in addition, implant denture does not need to be frequently taken off and worn, and is easy to clean, and has no foreign body sensation in use.

[0003] However, the implantation site needs to have sufficient bone width and bone height to maintain the stability of the implant. At the same time, the shape of the soft tissue also plays an important role in the aesthetics and function after implant repair. After tooth extraction, the general alveolar ridge often becomes narrow and flat, and the soft tissue also shrinks and collapses, which causes great difficulty for subsequent repair, especially implant repair. The concept of site preservation was first proposed in 1994, which refers to taking intervention measures on fresh extraction socket by using various methods and bone substitute materials to reduce bone loss of the extraction socket, promote bone regeneration, and maximize the retention of the original soft and hard tissue profile around the extraction socket.

[0004] At present, the main method for tooth extraction site preservation is to fill bone powder, which is to implant different artificial bone powders in the alveolar socket to promote new bone formation and reduce the absorption of alveolar bone width and height. However, bone powder filling has the disadvantages of early shedding of surface bone powder and rejection reaction of artificial bone powder, and the healing period of the alveolar socket filled with bone powder alone is long and needs to be improved. SUMMARY

[0005] In view of the problems in the prior art, the present application provides application of a plasmid composition in preparation of a drug for promoting healing of a tooth wound area.

[0006] The DNA plasmid composition comprising SEQ ID NO: 1 or the DNA plasmid composition comprising a sequence similar to SEQ ID NO: 1 with a similarity of more than 90% is used for the preparation of a drug for promoting tooth site preservation or healing of a tooth wound area.

[0007] In some embodiments, the site preservation or wound healing of the tooth site is in a dentition defect.

[0008] In some embodiments, the wound healing of the tooth site is site preservation.

[0009] In some embodiments, the site preservation of the tooth site is site preservation in a dentition defect.

[0010] In some embodiments, the dentition defect comprises caries, pulpitis, periapical periodontitis, periodontitis, gingivitis.

[0011] In some embodiments, the drug is used to repair at least one of (a) to (e) in the wound area of the tooth site:

[0012] (a) increase the bone volume fraction

[0013] (b) increase the trabecular bone number

[0014] (c) increase the bone density

[0015] (d) increase the average thickness of the trabecular bone

[0016] (e) reduce the trabecular bone separation degree.

[0017] The combination of the composition of the DNA plasmid containing the sequence of SEQ ID NO: 1 and the bone powder promotes site preservation or wound healing in a dentition defect.

[0018] In some embodiments, the drug dosage form of the DNA plasmid composition containing SEQ ID NO: 1 is an injection.

[0019] Preferably, the solvent in the injection is a 5% glucose solution.

[0020] Compared with the prior art, the advantages of the present application are:

[0021] The plasmid composition of the present application can shorten the alveolar bone repair and reconstruction time, maintain better bone density, alveolar ridge height and width; and the plasmid composition of the present application has no obvious local irritation, high safety, and has good application prospect.

[0022] At the same time, injecting the plasmid solution of the present application near the extraction socket and filling with bone powder will further improve the effect of site preservation. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is a graph of the body weight of each group of mice during the test time;

[0024] Figure 2 The figure is a graph of the food intake of each group of mice during the test period;

[0025] Figure 3 CT images of the tooth wound area of mice in each group at 14 days of the experiment;

[0026] Figure 4 CT images of the tooth wound area of mice in each group at 21 days of the experiment;

[0027] Figure 5 HE staining images of mice in each group at 14 days of the experiment;

[0028] Figure 6 HE staining images of mice in each group at 21 days of the experiment;

[0029] Figure 7 Bone parameter values of mice in each group at 14 days of the experiment (in the figure, A is A.BV / TV: relative bone volume fraction; B is Tb.Th: average thickness of bone trabecula; C is BMD: bone density; D is Tb.N: bone trabecula number; and E is Tb.Sp: bone trabecula separation degree.)

[0030] Figure 8 Bone parameter values of 21 mice in each group (in the figure, A is A.BV / TV: relative bone volume fraction; B is Tb.Th: average thickness of bone trabecula; C is BMD: bone density; D is Tb.N: bone trabecula number; and E is Tb.Sp: bone trabecula separation degree.)

[0031] Figure 9 Plasmid structure of the application. DETAILED DESCRIPTION

[0032] The application will be described in detail below in combination with the drawings and specific examples.

[0033] Test materials:

[0034] Bio-oss bone powder: production unit Geistlich Company

[0035] Physiological saline (specification: 100 mL: 0.9 g): production unit Guangdong Otsuka Pharmaceutical Co., Ltd.

[0036] Chloral hydrate: production unit: Shanghai Yuan Ye Biological Technology Co., Ltd.

[0037] EDTA (ethylene diamine tetraacetic acid) decalcification solution: production unit: Tianjin Zhonglian Chemical Reagent Co., Ltd.

[0038]

[0039] Example 1

[0040] Solution preparation

[0041] A solution of DNA plasmid containing the sequence of SEQ ID NO: 6 was prepared using a 5% glucose solution, and the specific plasmid structure is shown as follows: Figure 9 It was stored at -20 degrees Celsius. When administered, it was taken out and placed at 4 degrees Celsius for a period of time before administration.

[0042] Example 2

[0043] The test groups are as follows: Table 1

[0044]

[0045] (I) Establishment of tooth extraction wound model in C57BL / 6 mice

[0046] (1) 10% chloral hydrate anesthesia;

[0047] (2) Lying fixed on the operating table, the mouse's mouth was stretched open with rubber bands, and the oral cavity was disinfected with iodophor cotton balls;

[0048] (3) Separation of gingiva. The gingiva was separated along the right maxillary molar with a metal dental elevator to avoid tearing the gingiva during tooth extraction; the right maxillary first molar was loosened. The elevator was inserted into the right maxillary molar mesial to loosen it, avoiding the use of force to cause root fracture;

[0049] (4) Extraction of the right maxillary first molar. To prevent root fracture, the right maxillary first molar was loosened and then extracted with an ophthalmic forceps. The oral cavity was cleaned, the tooth extraction wound was stopped bleeding, and the mouse was prevented from suffocating due to accidental aspiration of blood. Bone powder was filled in all groups.

[0050] (5) After tooth extraction, the mouse was placed on a heating pad and returned to the cage after waking up;

[0051] (II) Administration

[0052] (1) Dose design: the administration dose of the test groups was grouped according to the groups shown in Table 1, a total of 3 groups, the control group was directly filled with bone powder, the vehicle group was injected with 80uL of 5% glucose injection and simultaneously filled with bone powder, and the administration group was injected with plasmid solution containing 40μg of plasmid;

[0053] (2) Administration route: administration was performed in a timely manner after tooth extraction, using mucosal injection.

[0054] (III) Test method

[0055] (1) Modeling: mice were randomly grouped by weight when their body weight reached about 20g, and the mice were anesthetized according to the group. The tooth extraction wound model mice were established.

[0056] (2) Submucosal injection of the tooth socket after tooth extraction on the first day.

[0057] (IV) Detection index

[0058] (I) General observation

[0059] The general observation of soft tissue healing was made. The mucosal tissue coverage of all defect areas and the wound healing of bone defect areas in each group were observed, and the presence or absence of infection was observed.

[0060] (2) Histological observation

[0061] HE staining: the growth of new bone in the tooth extraction wound, the level of inflammatory cell infiltration, and the arrangement of new bone trabeculae were observed.

[0062] (3) CBCT imaging analysis

[0063] The shape of the defect area, bone density, and bone parameters such as Tb.N (trabecular number), BV / TV (relative bone volume fraction), BMD (bone mineral density), Tb.Th (trabecular mean thickness), and Tb.Sp (trabecular separation) of new bone were displayed.

[0064] (V) Data collection and statistical analysis

[0065] One-way Anova was performed on the quantitative index. The results are expressed as , and the results with statistically significant differences are marked. "*" indicates P < 0.05 compared with the solvent group, with statistically significant differences; "**" indicates P < 0.01 compared with the solvent group, with statistically significant differences.

[0066] (VI) Test results

[0067] (1) Clinical observation results of the model

[0068] Intraoral phase during modeling: less blood at the tooth extraction wound site of the mouse.

[0069] 14-day intraoral phase of the mouse: the mucosa at the tooth extraction wound site of the mouse grew well without infection.

[0070] Maxillary tooth extraction wound healing process: two weeks after the extraction of the first maxillary molar in C57BL / 6 mice, the tooth extraction wound gradually recovered.

[0071] No mice died during the test period.

[0072] (2) Body weight and food intake

[0073] The changes in the body weight of the mice after tooth extraction are shown in Figure 1 , and the food intake of the mice is shown inFigure 2 As shown in the figure, the weight change and food intake of the mice in the administration group after tooth extraction had no obvious difference compared with the solvent group and the control group (P>0.05), indicating that after the mice were injected with the solution of the DNA plasmid containing the sequence of SEQ ID NO: 1, the mice did not experience sudden weight loss, and the safety was high and the side effects were low.

[0074] (3) Micro CT tooth extraction wound surface bone healing and situation

[0075] As shown in the figure, on the 14th day, the tooth extraction socket in the control group and the solvent group was relatively empty, and the bone was osteoporotic; the bone in the tooth socket of the administration group was filled. Figure 3

[0076] As shown in the figure, on the 21st day, the bone in the tooth socket of the mice in each group was more dense than on the 14th day. Compared with the control group and the solvent group, the administration group had better bone formation effect. Figure 4

[0077] (4) HE staining results of mouse tooth extraction wound bone trabecula

[0078] As shown in the figure, on the 14th day, fibrous tissue and osteoblasts were observed in the tooth extraction wounds of the control group and the solvent group, and osteoid was formed; the administration group had mature lamellar bone formation, mature bone trabecula, and restored normal arrangement structure. Figure 5 As shown in the figure, on the 21st day, the alveolar bone healing of the mice in each group of the tooth extraction wound healing model was basically completed. The bone marrow cavity of the control group and the solvent group was wide, while the bone marrow cavity of the administration group was narrower than that of the solvent group, the bone trabecula was connected to each other, and the arrangement was compact.

[0079] Figure 6 (5) Bone parameter analysis of each group of mice tooth extraction wound area

[0080] As shown in the figures, compared with the control group, the data of the solvent group had no significant difference, indicating that the use of 5% glucose solution as a solvent did not affect the healing of the tooth extraction wound area.

[0081] As shown in the figures, compared with the control group, the data of the relative bone volume fraction (BV / TV), the average bone trabecula thickness (Tb.Th), the bone mineral density (BMD), the bone trabecula number (Tb.N), and the bone trabecula separation degree (Tb.Sp) of the administration group had significant differences. It is indicated that the plasmid injection solution of the present application has obvious effect of improving the healing of the tooth wound area, and can effectively promote the growth of the alveolar bone. Figure 7 Figure 8

[0082] Compared with the control group, the data of the relative bone volume fraction (BV / TV), the average bone trabecula thickness (Tb.Th), the bone mineral density (BMD), the bone trabecula number (Tb.N), and the bone trabecula separation degree (Tb.Sp) of the administration group had significant differences. It is indicated that the plasmid injection solution of the present application has obvious effect of improving the healing of the tooth wound area, and can effectively promote the growth of the alveolar bone.

[0083] ​​​​​In summary, during the experiment, no mice died, there were no significant differences in body weight, food intake, and clinical symptoms among the groups, and no obvious abnormalities were observed at the local injection sites of the drug administration groups, indicating that the product has no obvious local irritation and systemic adverse reactions when injected submucosally near the tooth extraction socket, and has high safety.

[0084] The results of gross observation, CT scanning, and HE staining showed that, compared with the vehicle group, the tooth wound area was better in terms of bone parameters and osteogenesis after mucosal injection of the plasmid solution of the application. Therefore, after injection of the plasmid solution of the application, the alveolar bone repair and reconstruction time can be shortened, and better bone density, alveolar ridge height, and width can be maintained, which has a good application prospect.

[0085] Meanwhile, injecting the plasmid solution of the application submucosally near the tooth extraction socket and filling with bone powder will further improve the site preservation effect.

Claims

1. Use of a DNA plasmid composition comprising SEQ ID NO:1 or a DNA plasmid composition comprising having a sequence similarity of more than 90% to that of SEQ ID NO:1 in the preparation of a medicament for promoting healing of dental wounds.

2. The use according to claim 1, characterized in that, The healing of the dental wound area specifically refers to the preservation of the tooth site.

3. The use according to claim 2, characterized in that, The preservation of tooth sites specifically refers to the preservation of tooth sites in cases of tooth loss.

4. The use according to claim 3, characterized in that, The tooth loss is caused by dental caries, pulpitis, periapical periodontitis, periodontitis, or gingivitis.

5. The use according to claim 1, wherein the drug is used to repair at least one of (a) to (e) of the dental trauma area: (a) Increase bone volume fraction (b) Increase the number of trabeculae (c) Increase bone density (d) Increase the average thickness of trabecular bone (e) Reduce trabecular separation.

6. A composition comprising a DNA plasmid containing the sequence of SEQ ID NO:1, or a composition comprising a DNA plasmid having a sequence similarity of more than 90% to that of SEQ ID NO:1, and bone powder for the combined use of promoting site preservation in dentition defects.

7. The use according to any one of claims 1-6, characterized in that, The drug dosage form of the DNA plasmid composition containing SEQ ID NO:1 is an injection.

8. The use according to claim 7, characterized in that, The solvent in the injection is a 5% glucose solution.

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