CN103767881A - Dental pulp capping preparation and preparation method thereof - Google Patents

Abstract

The invention relates to the field of research and development of tissue regeneration materials, and discloses a tooth pulp capping agent and a preparation method thereof. The tooth pulp capping agent is rhBMP2-PLGA slow-release microspheres; the preparation method comprises the following steps: adding the first rhBMP2 solution into the PLGA dichloromethane solution, ultrasonically emulsify under ice bath conditions to form uniform colostrum, and use a high-speed homogenizer to stir at 10,000r/min; extract and stir the colostrum into the PVA solution at a uniform speed, and evenly disperse to form Double emulsion; magnetic stirring, centrifugation to collect the precipitate after volatilization of dichloromethane; repeated washing with deionized water and freeze-drying to obtain rhBMP2-PLGA sustained-release microspheres, which were stored at 4°C. The rhBMP2-PLGA slow-release microspheres of the present invention have slow-release and long-acting effects, protect the activity of related factors of gene recombinant human bone morphogenic protein 2, and form new dentin to achieve the purpose of protecting living pulp.

Description

A kind of tooth pulp capping agent and preparation method thereof

technical field

The invention relates to the field of research and development of tissue regeneration materials, in particular to a tooth pulp capping agent and a preparation method thereof.

Background technique

Pulp preservation is the most biological method in the treatment of pulp diseases, and the role of pulp capping agent is very important in its treatment. Among the many pulp capping materials currently used, bone morphogenetic protein (Bone Morphogenetic Protein, BMP) is one of the materials that has been widely concerned. It has been reported that BMP used as pulp capping treatment can form a large amount of restorative dentin to seal the exposed pulp hole in a short period of time, which is beneficial to the recovery of dental pulp. However, when BMP is used alone in the body, it will be diluted or destroyed by protease soon, and the action time on dental pulp cells is short, so it cannot effectively exert its due biological properties; moreover, it cannot provide a scaffold for dentin formation , making it very limited in clinical application.

Contents of the invention

The purpose of the present invention is to provide a kind of tooth pulp capping agent and preparation method thereof, use rhBMP2-PLGA slow-release microsphere material as tooth pulp capping agent, can solve the deficiency of BMP in application, can play slow-release effect; It can protect the activity of related factors of recombinant human bone morphogenetic protein-2 (rhBMP-2), and achieve the purpose of protecting living marrow.

In order to achieve the above object, the present invention adopts the following technical solutions to achieve.

A tooth pulp capping agent, characterized in that the tooth pulp capping agent is rhBMP2-PLGA slow-release microspheres.

The preparation method of the above dental pulp capping agent is characterized in that it comprises the following steps: adding the first rhBMP2 solution to the PLGA dichloromethane solution, ultrasonic emulsification under ice bath conditions to form uniform colostrum, and using a high-speed homogenizer at 10000r/m Stir under the condition of min; extract and stir the colostrum and inject it into the PVA solution at a uniform speed, and evenly disperse to form a double emulsion; magnetically stir to make the dichloromethane volatilize completely, then centrifuge to collect the precipitate; freeze-dry after repeated washing with deionized water, The rhBMP2-PLGA slow-release microspheres used as dental pulp capping agent were obtained and stored at 4°C.

In the above technical scheme, preferably, the mass-volume concentration of the rhBMP2 solution is 0.1% (mg/ml), the mass-volume concentration of the PLGA dichloromethane solution is 2.5% (mg/ml), and the mass-volume concentration of the PVA solution It is 1% (mg/ml), and the volume ratio of rhBMP2 solution, PLGA dichloromethane solution, and PVA solution is 1:10:100.

The present invention uses rhBMP2-PLGA slow-release microspheres as dental pulp capping agent, recombinant human bone morphogenetic protein-2 (rhBMP-2) as the main body, and polylactic acid-glycolic acid copolymer [poly( lactic-co-glycolic acid), PLGA] as a scaffold, which solves the technical problem that rhBMP-2 is rapidly diluted and destroyed by protease in vivo, has slow-release and long-acting effect, and protects recombinant human bone morphogenetic protein 2 (recombinant human bonemorphogenetic protein -2 (rhBMP-2)) to form new dentin to achieve the purpose of protecting the living pulp.

In addition, PLGA has good biocompatibility, biodegradability, and non-toxicity; and its own and its degradation products have no obvious adverse reactions to polypeptides, proteins, etc., safe and reliable, and has been approved by the US FDA for clinical use.

Description of drawings

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

Figure 1 is a scanning electron micrograph of rhBMP2-PLGA sustained-release microspheres.

Figure 2 is a particle size distribution diagram of rhBMP2-PLGA sustained-release microspheres.

Fig. 3 is a broken line graph of the in vitro release of rhBMP2-PLGA sustained-release microspheres.

Figure 4 is a diagram of the experimental results of the blank control group, in which there is no hard tissue formation.

Figure 5 is the experimental results of the rhBMP2 group, in which there are dentin calcification bridges, but relatively few.

Figure 6 is the experimental results of the rhBMP2-PLGA sustained-release microsphere group, in which dentin was formed and a complete calcified bridge was formed.

Figure 7 is a diagram of the experimental results of the collagen group, in which complete dentin bridge formation can be observed;

Figure 8 is the experimental results of the antibiotic group, in which calcified hard tissue was formed, but there was no complete dentin calcification bridge.

Figure 9 is a picture of the experimental results of the mixed group of antibiotics and rhBMP2-PLGA sustained-release microspheres, in which a complete dentin calcification bridge can be observed.

Figure 10 is the experimental results of the mixed group of collagen and rhBMP2-PLGA sustained-release microspheres. In the figure, it can be observed that a complete dentin calcification bridge is formed, and there is no calcification in the pulp cavity.

Fig. 11 is a histogram of dentin bridge thickness in various animal experiments.

Detailed ways

In the present invention, PLGA and PVA were purchased from SIGMA Company; rhBMP2 was purchased from Shanghai Ruibang Biomaterials Co., Ltd.; rhBMP2 ELISA detection kit was purchased from Wuhan Boster Company. The rhBMP2-PLGA sustained-release microspheres were prepared by (oil-in-water, water-in-oil) W/O/W double emulsion solvent evaporation method.

1. Experimental method

(1) Experimental preparation:

Add 200ul of rhBMP2 solution containing 0.1% (mg/ml) mass-volume concentration (inner water phase, set as W1) to 2ml of PLGA dichloromethane solution containing 2.5% (mg/ml) mass-volume concentration (oil phase , set as O), ultrasonically emulsify in ice bath to form uniform colostrum (W1/O), immediately use a high-speed homogenizer to stir at 10000r/min; use a syringe to extract the stirred colostrum slowly and uniformly Inject into 20ml of PVA solution containing 1% (mg/ml) mass-volume concentration (outer water phase, set as W2), and disperse evenly for 2 minutes to form W1/O/W2 double emulsion; magnetically stir for 3 to 4 hours, so that After dichloromethane volatilized completely, centrifuge at 15,000 r/min for 5 min to collect the precipitate; wash with deionized water for 5 times, freeze-dry for 48 h to obtain rhBMP2-PLGA sustained-release microspheres, and store at 4°C.

(2) Morphological observation:

Take an appropriate amount of rhBMP2-PLGA sustained-release microspheres and evenly disperse them on a metal plate with double-sided adhesive tape, spray gold and observe its surface morphology with a scanning electron microscope (SEM).

(3) Determination of particle size and distribution of microspheres:

Take an appropriate amount of rhBMP2-PLGA sustained-release microspheres and disperse them in ultrapure water. After the dispersion is uniform by ultrasonic oscillation, the distribution and particle size are measured in a laser particle size analyzer (Malvern Nano ZS-90).

(4) Determination of encapsulation efficiency and drug loading:

Accurately weigh 20 mg of rhBMP2-PLGA sustained-release microspheres, add 2 mL of 0.1mol/L NaOH (containing 0.5% SDS) solution, and incubate in a constant temperature water bath at 37°C for 24 hours with shaking to completely dissolve the microspheres; neutralize with 0.1mol/L HCl After reaching a pH value of 7.0, centrifuge at 12,000 r/min for 5 min, take the supernatant, and use ELISA to measure the content of rhBMP2 in the drug-loaded microspheres. The specific operation is carried out according to the instructions on the kit. And calculate encapsulation efficiency and drug loading capacity according to the following formulas respectively:

Encapsulation rate of microspheres (%) = (drug content in microspheres / dosage) × 100%

Drug loading of microspheres (%) = (drug content in microspheres/mass of microspheres) × 100%

(5) Calculate the release amount:

Accurately measure 10 mg of rhBMP2-PLGA sustained-release microspheres dissolved in 5 ml of PBS buffer (20 mmol) pH7.4 as the release medium, shake at 37 ° C, and take out the supernatant at 1, 3, 7, 14, and 28 days respectively 200ul and add a fresh equal amount of release medium. The concentration of rhBMP2 in the supernatant was measured by rhBMP2ELISA, and the released amount was calculated.

(6) Animal experiments:

Select 2 healthy adult beagle dogs (purchased from Xi'an Dilepu Biological Resources Development Co., Ltd.), SPF level (safety and health level), weighing 10.8kg and 11.2kg respectively, aged 12-14 months, both male , the dentition is complete and the periodontium of the teeth is healthy, and there is no disease in the whole body. Before the experiment, they were adaptively fed for 1 to 2 weeks, and they were used for the experiment after the temperament was stable.

Each dog selected premolars and incisors on both sides, a total of 42 teeth, and randomly divided the 42 teeth into 7 groups according to the random number table method: rhBMP2 group, rhBMP2-PLGA sustained-release microspheres, collagen, dental antibiotics, dental antibiotics﹢ rhBMP2-PLGA sustained-release microspheres, collagen + rhBMP2-PLGA sustained-release microspheres, blank control group; 6 teeth in each group. Animals were sacrificed after feeding for 3 months after the experiment.

Fasting began 12 hours before the pulp capping operation. Preoperative intramuscular injection anesthesia with Sumianxin injection at a dose of 0.1ml/Kg 30 minutes before the operation, and additionally inject pentobarbital sodium with a mass-volume concentration of 3% during the operation according to the anesthesia situation to maintain the anesthesia effect . After anesthesia, the experimental dog was fixed supine on the operating table, the oral cavity was rinsed with 3% hydrogen peroxide solution, and the oral cavity and experimental teeth were disinfected with 1% iodine tincture with a mass-volume concentration and 75% alcohol with a volume concentration. On the occlusal surface of the experimental teeth, use a high-speed turbine handpiece ball drill to expose the pulp with a diameter of 1mm, gently rinse with 10ml of sodium hypochlorite with a mass-volume concentration of 2.5%, and then use 2ml of EDTA with a mass-volume concentration of 17% (Chinese name: ethylenediamine) Tetraacetic acid ) rinse, stop bleeding after rinsing, dry, put pulp capping agent on the exposed pulp, put gelatin sponge with basically the same size, and fill with glass ionomer. The specific operation steps are as follows:

Blank control group: place gelatin sponge directly, fill with glass ionomer, and apply vaseline to prevent moisture.

rhBMP2 group: rhBMP2 was soaked in gelatin sponge and placed directly on the pulp section, filled with glass ionomer, and coated with vaseline to prevent moisture.

rhBMP2-PLGA sustained-release microspheres group: place rhBMP2-PLGA sustained-release microspheres directly on the pulp section, place gelatin sponge, fill with glass ionomers, and apply vaseline to prevent moisture.

Collagen group: Use a gelatin sponge to dip an appropriate amount of collagen and place it on the pulp section, fill it with glass ionomers, and apply Vaseline to prevent moisture.

Antibiotic group: mix the two preparations evenly, soak an appropriate amount with a gelatin sponge and place it on the pulp section, fill it with glass ionomers, and apply vaseline to prevent moisture.

Mixed group of antibiotics and rhBMP2-PLGA sustained-release microspheres: a gelatin sponge soaked with an appropriate amount of antibiotics was dipped in an appropriate amount of white powder of rhBMP2-PLGA sustained-release microspheres (after freeze-drying), placed on the pulp section, and filled with glass ionomers , and apply vaseline to prevent moisture.

Collagen and rhBMP2-PLGA sustained-release microspheres mixed group: Dip a gelatin sponge soaked with an appropriate amount of collagen to take an appropriate amount of white powder of rhBMP2-PLGA sustained-release microspheres, place it on the pulp section, fill it with glass ion, and apply Vaseline septum wet.

In this experimental example, the antibiotic is a mixture of equal parts of metronidazole, ciprofloxacin, minocycline, and minocycline; and the whole operation process is completed by the same person, and the animals eat soft food for the first three days after operation. , antibiotic injection for three days. After the operation, the condition of the stuffing in the oral cavity and the general mental state of the experimental dogs were observed regularly. X-ray films were taken of the experimental teeth after operation. Three months after the operation, the animals were sacrificed, and the hard tissue formation was observed by MicroCT scanning.

2. Experimental results

(1) The rhBMP2-PLGA sustained-release microspheres prepared under the optimized process were in the form of white powder after lyophilization without collapse; the rhBMP2-PLGA sustained-release microspheres were round and uniform in shape, smooth in surface, and free of adhesion, as shown in Figure 1.

(2) The particle size distribution of rhBMP2-PLGA slow-release microspheres is normal distribution, as shown in Figure 2, the distribution range is concentrated, and the particle size distribution of most microspheres is between 3um and 4um.

(3) The encapsulation efficiency of rhBMP2-PLGA sustained-release microspheres was 72.5%±4.17%, and the drug loading rate of the microspheres was 0.38%±0.24%. The release rate of the sustained-release microspheres loaded with rhBMP2 was 19.36%±1.83% when released in vitro for 1 day, showing a burst release, which was mainly due to the drug release on the surface of the sustained-release microspheres, and the subsequent release was relatively slow, but the cumulative The release amount continued to increase and reached 52.76%±3.7% at 28 days. As shown in Figure 3.

(4) Animal experiment results: MicroCT observed and measured the thickness of new dentin along the labial-lingual to tooth long axis of the experimental teeth, and calculated the average value of each group.

Blank control group: as shown in Figure 4, no hard tissue formation was observed;

rhBMP2 group: As shown in Figure 5, complete dentin calcification bridges can be observed, but relatively few.

rhBMP2-PLGA slow-release microsphere group: as shown in Figure 6, it can be observed that a complete calcification bridge is formed, and there is no calcification in the medullary cavity;

Collagen group: as shown in Figure 7, complete dentin bridge formation can be observed;

Antibiotic group: as shown in Figure 8, calcified hard tissue can be observed, but there is no complete dentin calcification bridge;

Mixed group of antibiotics and rhBMP2-PLGA sustained-release microspheres: as shown in Figure 9, it can be observed that a complete dentin calcification bridge is formed, and there is no calcification in the pulp cavity;

Collagen and rhBMP2-PLGA slow-release microspheres mixed group: as shown in Figure 10, it can be observed that a complete dentin calcification bridge is formed, and there is no calcification in the pulp cavity.

As shown in Figure 11, there were statistically significant differences between the rhBMP2 group and the collagen group, the rhBMP2-PLGA sustained-release microspheres group, the mixed group of antibiotics and rhBMP2-PLGA sustained-release microspheres, and the mixed group of antibiotics and rhBMP2-PLGA sustained-release microspheres. difference (P<0.05).

3. Experimental conclusion

In this experiment, by comparing the results of using rhBMP2 without carrier with the experimental results of other groups, it was found that using rhBMP2 alone has a certain effect on inducing the formation of restorative dentin, but its effect is far less than that of using rhBMP2-PLGA sustained-release microspheres The other groups, the specific reason is that the organic substances in the pulp tissue have damaged the activity of rhBMP2.

The results of this experiment showed that rhBMP2-PLGA sustained-release microspheres and composite dental antibiotics had a stronger induction and differentiation effect on dental pulp cells after pulp capping than other groups, and the dentin bridge formed in the same time period was thicker and more complete. Good pulp capping effect provides feasibility for its clinical application.

Those skilled in the art can learn from the content of this article, appropriately improve the process parameters to realize, or make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention, to realize and apply the technology of the present invention. In particular, it should be pointed out that all the above-mentioned similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the protection scope of the present invention.

Claims (3)

1. A tooth pulp capping agent, characterized in that, the tooth pulp capping agent is rhBMP2-PLGA slow-release microspheres. 2. the preparation method of tooth pulp capping agent as claimed in claim 1, is characterized in that, comprises the following steps: first rhBMP2 solution is joined in PLGA dichloromethane solution, ultrasonic emulsification forms uniform colostrum under ice-bath condition, And use a high-speed homogenizer to stir under the condition of 10000r/min; extract the stirred colostrum and inject it into the PVA solution at a uniform speed, and evenly disperse to form a double emulsion; magnetically stir to make the dichloromethane volatilize completely, and then centrifuge to collect the sediment; The rhBMP2-PLGA slow-release microspheres used as tooth pulp capping agent were obtained after repeated washing with deionized water, and stored at 4°C. 3. the preparation method of tooth pulp capping agent as claimed in claim 2 is characterized in that, the mass-volume concentration of rhBMP2 solution is 0.1% (mg/ml), the mass-volume concentration of PLGA dichloromethane solution is 2.5% (mg/ml), the mass-volume concentration of PVA solution is 1% (mg/ml), and the volume ratio of rhBMP2 solution, PLGA dichloromethane solution, and PVA solution is 1:10:100.

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