CN112190599A

CN112190599A - Method for preparing composite sponge for treating dry socket syndrome

Info

Publication number: CN112190599A
Application number: CN202011072003.8A
Authority: CN
Inventors: 姜力群; 崔飞艳; 孟箭; 王子尧; 杨玲; 张咪; 王云
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2020-10-09
Filing date: 2020-10-09
Publication date: 2021-01-08
Anticipated expiration: 2040-10-09
Also published as: CN112190599B

Abstract

The invention discloses a method for preparing a composite sponge for treating dry socket syndrome, which comprises the steps of mixing a staphylococcus aureus bacterial wall with a sodium alginate solution, pre-freezing, and freeze-drying in a freeze dryer to prepare the staphylococcus aureus bacterial wall sponge; preparing mixed solution of gelatin, chitosan and calcium lactate; and adding the mixed solution into staphylococcus aureus bacteria mural sponge, pre-freezing and freeze-drying. According to the invention, the typing change of immune cells after bacterial infection is utilized, the bacterial wall of staphylococcus aureus is adopted, and chitosan, gelatin and sodium alginate are used as matrixes to construct the composite sponge, and researches show that the composite sponge prepared by the invention can effectively regulate and control local immune cells such as macrophage and the like, can convert the local immune cells into typing for promoting tissue repair, and can effectively avoid inflammatory reaction caused by bacterial infection, so that the composite sponge has a good treatment effect on dry socket syndrome, and has a good clinical application value.

Description

Method for preparing composite sponge for treating dry socket syndrome

Technical Field

The invention belongs to the technical field of dry socket disease treatment, and particularly relates to a method for preparing a composite sponge for treating dry socket disease.

Background

The dry socket is one of the common complications after the dental surgery extraction, the posterior teeth of the lower jaw are common, particularly after the third molar extraction of the lower jaw impacted patient, under the normal condition, even if the third molar extraction of the lower jaw is a flap bone removal extraction operation, the pain of the wound can gradually disappear after 2 to 3 days, if severe pain appears after 2 to 3 days after the tooth extraction, the pain radiates to the temporal part of the ear, the lower jaw area or the top of the head, and can not be relieved by using a common analgesic, so that the dry socket can be generated. The clinical examination shows that the alveolus is empty and deficient or has putrefactive blood clots which are grey white. The broken objects covered on the alveolar fossa wall have odor, and the probe can directly touch the bone surface and have sharp pain. There is no obvious swelling on the jaw and face, no obvious restriction on opening, and swollen lymph nodes and tenderness under the mandible.

Conventionally, an iodoform gauze is adopted to prevent wound infection, but the iodoform gauze has the problems of slow wound healing, need to take out the gauze again and the like. Therefore, how to retain the infection prevention effect of the iodoform sliver, quickly promote wound healing and avoid the problem of taking out again is the problem to be solved for treating dry groove.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The invention provides a method for preparing a composite sponge for treating dry socket syndrome.

In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing a composite sponge for treating dry socket disease comprises the following steps,

mixing the staphylococcus aureus bacterial wall with a sodium alginate solution, pre-freezing, and freeze-drying in a freeze dryer to prepare a staphylococcus aureus bacterial wall sponge;

preparing mixed solution of gelatin, chitosan and calcium lactate;

and adding the mixed solution into staphylococcus aureus bacteria mural sponge, pre-freezing and freeze-drying.

As a preferred embodiment of the method for preparing the composite sponge for treating dry socket syndrome, the method comprises the following steps: the staphylococcus aureus bacterial wall is mixed with the sodium alginate solution, wherein the mass concentration of the staphylococcus aureus bacterial wall in the mixed solution is 0.1% -2%, and the mass concentration of the sodium alginate aqueous solution is 2.5%.

As a preferred embodiment of the method for preparing the composite sponge for treating dry socket syndrome, the method comprises the following steps: preparing gelatin, chitosan and calcium lactate into a mixed solution; the preparation method comprises mixing 1% gelatin, 1% chitosan, and 0.5% calcium lactate to obtain mixed water solution.

As a preferred embodiment of the method for preparing the composite sponge for treating dry socket syndrome, the method comprises the following steps: the reaction was carried out for 30 min.

As a preferred embodiment of the method for preparing the composite sponge for treating dry socket syndrome, the method comprises the following steps: adding the mixed solution into a sodium alginate sponge, namely adding a mixed aqueous solution prepared from 1% of gelatin, 1% of chitosan and 0.5% of calcium lactate into a staphylococcus aureus bacteria wall sponge, reacting for 30min, and freeze-drying, wherein the ratio of the mixed solution of 1% of gelatin, 1% of chitosan and 0.5% of calcium lactate to the staphylococcus aureus bacteria wall sponge is as follows: the dry weight ratio is 1: 1.

As a preferred embodiment of the method for preparing the composite sponge for treating dry socket syndrome, the method comprises the following steps: the preparation method of the staphylococcus aureus bacterial wall comprises the steps of inoculating staphylococcus aureus into a liquid culture medium for culturing for 48 hours, sub-packaging a liquid culture medium suspension containing bacteria into a centrifugal tube, centrifuging for 20min at 12000rm, discarding supernatant, taking precipitate, and using 10mMgSO (magnesium sulfate) in the precipitate₄Washing with 10mM Tris-HCl solution (pH 7.4), centrifuging at 12000rm for 20min, discarding the supernatant, and adding 10mM MgSO₄The 10mM Tris-HCl solution with the pH value of 7.4 is poured into a beaker, the mixture is crushed by ultrasound for 20min, the suspension is subpackaged in a centrifuge tube, the centrifugation is carried out for 2000rm5min, the supernatant is taken, 12000rm is taken, the centrifugation is carried out for 20min, the precipitate is taken, the mixture is put into a centrifuge tube, the 12000rm is taken, the centrifugation is carried out for 20min, and the freeze-drying is carried out.

The invention has the beneficial effects that: according to the invention, the typing change of immune cells after bacterial infection is utilized, the bacterial wall of staphylococcus aureus is adopted, and chitosan, gelatin and sodium alginate are used as matrixes to construct the composite sponge, and researches show that the composite sponge prepared by the invention can effectively regulate and control local immune cells such as macrophage and the like, can convert the local immune cells into typing for promoting tissue repair, and can effectively avoid inflammatory reaction caused by bacterial infection, so that the composite sponge has a good treatment effect on dry socket syndrome, and has a good clinical application value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a diagram of dry socket syndrome in rats, and the red area is the condition of tooth extraction wound infection.

FIG. 2 is a comparison of soft tissue recovery at days 7 and 14 for the placebo group, the bacterial wall sponge group (group A), the bacteria free wall sponge group (group B), and the iodoform sliver group (group C).

FIG. 3 shows the results of Elisa examination of the local tissues of a blank control group, a bacterial wall sponge group (group A), a bacterial wall free sponge group (group B) and an iodoform yarn group (group C).

FIG. 4 shows the results of PCR examination of cells after incubation of bacterial wall sponges (group A), non-bacterial wall sponges (group B), iodoform strips (group C) with mouse RAW264.7 cells for 24 h.

FIG. 5 shows HE staining results of alveolar socket tissue after treatment with bacterial mural sponge and blank control.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Experimental animals: 60 male SD rats, weighing approximately 140-. The protocol was approved by the animal ethics committee of xu university of medical.

Grouping experimental drugs: A. bacteria wall composite sponge: preparing 24mL of staphylococcus aureus bacterial wall with mass concentration (g/mL) of 0.5% and 2.5% sodium alginate aqueous solution, adding into a 48-pore plate, wherein each pore is 0.5mL, pre-freezing at-20 ℃, and freeze-drying in a freeze dryer; mixing gelatin with a mass concentration (g/mL) of 1%, chitosan with a mass concentration of 1% and calcium lactate with a mass concentration of 0.5% to obtain a total of 24mL, adding into Staphylococcus aureus bacterial wall and sodium alginate sponge with a mass concentration of 0.5mL per sponge, reacting for 30min, pre-freezing at-20 deg.C, and lyophilizing. B. Bacteria-free wall sponges: 2.5 percent sodium alginate solution, pre-freezing at-20 degrees, and freeze-drying in a freeze dryer; preparing mixed solution of 1% gelatin, 1% chitosan, and 0.5% calcium lactate, adding into sponge of sodium alginate, reacting for 30min, pre-freezing at-20 deg.C, and lyophilizing. C. An iodoform sliver.

The preparation method of the 0.5 percent staphylococcus aureus bacterial wall comprises the following steps: staphylococcus aureus (ATCC6538) was inoculated in a liquid medium (1X 10 inoculum per liter)⁹cfu) for 48h, sub-packaging the liquid medium suspension containing bacteria in a centrifuge tube, centrifuging at 12000rm for 20min, discarding supernatant, collecting precipitate, and adding 10mMgSO₄Washing with 10mM Tris-HCl solution (pH 7.4), centrifuging at 12000rm for 20min, discarding the supernatant, and adding 10mM MgSO₄The 10mM Tris-HCl solution with the pH value of 7.4 is poured into a beaker, the mixture is crushed by ultrasound for 20min, the suspension is subpackaged in a centrifuge tube and centrifuged for 2000rm5min, the supernatant is taken and then 12000rm is taken and centrifuged for 20min, the precipitate is taken and put into a centrifuge tube and then 12000rm is taken and centrifuged for 20min, and the mixture is freeze-dried. 0.5g of the freeze-dried product is re-dissolved in 100mL of deionized water, thus obtaining a 0.5% staphylococcus aureus bacterial wall solution.

Establishing a rat dry socket syndrome model: SD rats were intraperitoneally injected with 4% chloral hydrate (1ml/100g), after anesthesia, gums were separated with a probe, the first left and right molars of the upper jaw were raised, and alveolar bone walls were scratched to break, and wounds were enlarged. The tampon soaked with 1:1000 adrenaline hydrochloride is placed into the tooth extraction socket for 1-2min to contract blood vessels in the tooth extraction socket, and after blood seepage is stopped, the tampon dipped with saturated staphylococcus aureus strains is placed into the tooth extraction socket for 2 min. Returning the animals to the animal room after the animals revive, and feeding the animals by a conventional method after water is cut off for 0.5 h. After 3-4 days of infection, the wound socket of the extracted tooth of the rat is covered by a yellow low-grey necrotic tissue layer, the surrounding mucosa is red and swollen, and the wound is not obviously reduced, namely the model infection is successful.

Treatment: on the fourth day, rats were randomly divided into 3 groups of 20 rats each, group a using bacterial wall sponges, group B using non-bacterial wall sponges, and group C using iodoform strips. One side of the rat is used as a blank group and is not treated, the other side of the rat is used as a treatment group, putrefactive necrotic tissues are removed, cotton balls soaked with 3% hydrogen peroxide solution and cotton balls soaked with normal saline are used for alternately cleaning alveolus until the alveolus is clean and free from peculiar smell, and then medicines are put into the rat according to groups. After 3 days, the condition of the drug in the wound is checked, and if the drug falls off, the treatment is carried out again. Taking out the iodoform sliver at 1 week.

Observation indexes are as follows: 1. tooth extraction area measurement: the healing of the wound was visually observed and recorded at 7d, 10d and 14d after treatment, the maximum length (L) and width (W) of the wound were measured, the average was taken three times, and the change of the wound area S-L-W/(L-W)₀*W₀) 100%. The wound area change was observed to compare the soft tissue recovery of each group.

He staining: fixing with 4% paraformaldehyde for 2-3 days, decalcifying with 10% EDTA decalcifying liquid for 2-3 weeks, and replacing the decalcifying liquid for 2-3 days, wherein the decalcifying liquid is semitransparent and elastic in hand feeling, and the probe can smoothly penetrate into bone tissue to complete decalcification. Paraffin embedding, sagittal serial sections of 5 μm thickness along the long axis of the jaw bone, and three substantially identical positions for each set of sections were taken. The histological changes of the alveolar fossa were observed under a light microscope.

Elisa assay: after 14 days of treatment in the keratitis model, the local tissue was homogenized, centrifuged at 1000rpm for 20 minutes and the supernatant was removed. Taking out an Elisa kit (enzyme-labeled organism), re-warming for 20 minutes at room temperature, adding 50 mu L of standard substances with different concentrations into a pore plate, diluting the sample by 4 times by using a sample diluent, adding the diluted sample into the pore plate, adding 100 mu L of detection antibody marked by horseradish peroxidase (HRP) into each of standard substance pores and sample pores except blank pores, sealing reaction pores by using a sealing plate membrane, incubating for 60 minutes at 37 ℃ in a constant temperature box, discarding liquid after incubation is finished, patting the liquid on absorbent paper, fully filling cleaning solution diluted by 20 times into each pore, standing for 1 minute, throwing off the cleaning solution, patting the liquid on the absorbent paper, repeatedly washing the plate for 5 times, finishing plate washing, adding 50 mu L of substrate A, B into each pore, incubating for 15 minutes at 37 ℃ in a dark place, finally adding 500 mu L of stop solution into each pore, and measuring the OD value of each pore at a wavelength of 450nm within 15 minutes.

PCR assay: mouse RAW264.7 macrophage cells were inoculated in 6-well plates, bacterial wall complex sponges, bacteria-free wall sponges and iodoform gauze were incubated with the macrophage cells for 24h, respectively, with 0.1g of sponge or gauze added per well. After 24h, adding a proper amount of RNAiSiplus, blowing out the lysed cells, standing for 5 minutes, adding chloroform with the volume of 1/5 lysate, shaking, uniformly mixing, standing for 5 minutes, and centrifuging at 12000g at 4 ℃ for 15 minutes. And (2) adding isopropanol with the volume of 0.5-1 time of the lysate into the supernatant, gently mixing the mixture uniformly, standing the mixture at room temperature for 10 minutes, centrifuging the mixture at 12000g at 4 ℃ for 10 minutes, taking the precipitate, washing the precipitate by using 75% ethanol with the same volume as the lysate, centrifuging the precipitate at 7500g at 4 ℃ for 5 minutes, removing the supernatant, taking the precipitate, and drying the precipitate at room temperature to obtain the RNA extract. Dissolving the RNA extract in a certain amount of ribozyme-free water, measuring the RNA concentration under an RNA concentration tester, preparing a reverse transcription reaction solution according to the instructions of a reverse transcription kit, adding a sample containing a certain amount of RNA, uniformly mixing, and synthesizing cDNA in an RNA reverse transcription synthesizer under the conditions of reaction at 37 ℃ for 15 minutes, 85 ℃, 5 seconds and 4 ℃ for one cycle. Adding cDNA sample, primer, fluorescent solution and ribozyme-free water into a 96-well plate according to a 10-microliter volume system, sealing the plate with a membrane, centrifuging at 3500rpm and 4 ℃ for 5 minutes, and detecting the expression of each group of cytokines by using a PCR detector. The primer sequences are as follows:

and (5) observing the result by naked eyes: on day 4 after tooth extraction, the surface of the alveolar fossa was visually covered with a grayish yellow necrotic tissue layer, and the surrounding gingiva was red and swollen, and bleeding was detected (fig. 1, rat dry socket, red zone was the condition of infection of the tooth extraction wound). The rats had a slightly poor overall condition, reduced hair shine compared to before, and slightly reduced food intake.

Post-treatment observation, fig. 2 shows: the blank control group still has larger wound surface, the inflammation of the wound of the treatment group is reduced, the wound is gradually healed along with time, and the area is reduced. At 7 days, the blank control group healed slowly, the area of the tooth extraction wound was still large, and the healing rate among the three treatment groups was bacterial wall sponge (group a) > bacteria-free wall sponge group (group B) > iodoform yarn group (group C). At 14 days, the blank control group still had an obvious wound surface, the wound surface of the bacterial wall sponge group was almost completely healed, granulation tissue was visible in the tooth extraction wound, and the wound surface of the bacteria-free wall sponge group and the iodoform gauze group still remained unhealed.

TABLE 1 dental extraction area measurement

FIG. 3 shows Elisa results of 14 days of dry socket syndrome. Results show that compared with iodoform yarn and a group of sponge without bacterial wall, the bacterial wall composite sponge obviously improves the protein level of inflammation inhibiting factors (IL-10 and ARG-1) at dry channel parts, reduces the protein level of proinflammatory factors (TNF-alpha and iNOs) thereof, and shows that the bacterial wall composite sponge has good inflammation inhibiting effect on dry channel.

FIG. 4 shows the results of PCR examination of cells after incubating sponges with mouse RAW264.7 macrophages for 24 h. The results showed that both the bacterial wall complex sponge group (group a) and the bacteria-free wall sponge group (group B) significantly increased the gene expression levels of the anti-inflammatory factors (IL-10 and ARG-1) of RAW264.7 cells compared to the iodoform sliver group (group C), wherein both the bacterial wall complex sponge group were significantly higher than the bacteria-free wall sponge group. While the bacterial wall complex sponge group had significantly lower pro-inflammatory factor (TNF-a and iNOs) RNA expression than the remaining three groups. The results show that the bacterial wall composite sponge can improve the RNA expression of the macrophage anti-inflammatory factor and reduce the RNA expression of the proinflammatory factor by changing the macrophage typing so as to play a role in treating the dry socket disease.

FIG. 5 shows HE staining results of alveolar tissues of a bacterial mural sponge group and a blank control group, wherein the blank control group has severe inflammation at 1 week, a large number of neutrophils are arranged in an alveolar pit, lymphocyte infiltration is carried out, and inflammatory exudation is carried out on the surface. The continuity of the epithelium of the compound sponge group is basically recovered, the inflammation is obviously reduced compared with that of a blank control group, the range is reduced, and the fibrous connective tissue hyperplasia begins to appear around the compound sponge group. The continuity of the mucosa epithelium of the blank control group is not completely recovered at 2 weeks, while the epithelium of the compound sponge group is complete, the inflammation is more limited, and the fibrous connective tissue in the alveolus is proliferated.

According to the invention, the typing change of immune cells after bacterial infection is utilized, and the composite sponge is prepared by adopting a staphylococcus aureus bacterial wall, and researches show that the composite sponge prepared by the invention can effectively regulate and control local immune cells such as macrophage and the like, can be transformed to typing for promoting tissue repair, and can effectively avoid inflammatory reaction caused by bacterial infection, so that the composite sponge has a good treatment effect on dry socket syndrome, and has good clinical application value.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A method for preparing a composite sponge for treating dry socket syndrome is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

preparing mixed solution of gelatin, chitosan and calcium lactate;

2. The method of preparing a composite sponge for the treatment of dry socket according to claim 1, wherein: the staphylococcus aureus bacterial wall is mixed with the sodium alginate solution, wherein the mass concentration of the staphylococcus aureus bacterial wall in the mixed solution is 0.1% -2%, and the mass concentration of the sodium alginate aqueous solution is 2.5%.

3. The method for preparing a composite sponge for treating dry socket according to claim 1 or 2, wherein: preparing gelatin, chitosan and calcium lactate into a mixed solution; the preparation method comprises mixing 1% gelatin, 1% chitosan, and 0.5% calcium lactate to obtain mixed water solution.

4. The method for preparing a composite sponge for treating dry socket according to claim 1 or 2, wherein: the reaction was carried out for 30 min.

5. The method for preparing a composite sponge for treating dry socket according to claim 1 or 2, wherein: adding the mixed solution into a sodium alginate sponge, namely adding a mixed aqueous solution prepared from 1% of gelatin, 1% of chitosan and 0.5% of calcium lactate into a staphylococcus aureus bacteria wall sponge, reacting for 30min, and freeze-drying, wherein the ratio of the mixed solution of 1% of gelatin, 1% of chitosan and 0.5% of calcium lactate to the staphylococcus aureus bacteria wall sponge is as follows: the dry weight ratio is 1: 1.

6. The method for preparing a composite sponge for treating dry socket according to claim 1 or 2, wherein: the preparation method of the staphylococcus aureus bacterial wall comprises the steps of inoculating staphylococcus aureus into a liquid culture medium for culturing for 48 hours, sub-packaging a liquid culture medium suspension containing bacteria into a centrifugal tube, centrifuging for 20min at 12000rm, discarding supernatant, taking precipitate, and using 10mMgSO (magnesium sulfate) in the precipitate₄Washing with 10mM Tris-HCl solution (pH 7.4), centrifuging at 12000rm for 20min, discarding the supernatant, and adding 10mM MgSO₄The 10mM Tris-HCl solution with the pH value of 7.4 is poured into a beaker, the mixture is crushed by ultrasound for 20min, the suspension is subpackaged in a centrifuge tube, the centrifugation is carried out for 2000rm5min, the supernatant is taken, 12000rm is taken, the centrifugation is carried out for 20min, the precipitate is taken, the mixture is put into a centrifuge tube, the 12000rm is taken, the centrifugation is carried out for 20min, and the freeze-drying is carried out.