Sustained drug delivery methods for wound healing using silk hydrogels and products thereof

Abstract

The present invention provides a silk fibroin-based hydrogel and antibiotic composition comprising 5% silk fibroin embedded with 0.4-2% antibiotic, more particularly tetracycline or doxycycline, the antibiotic used to prepare the silk fibroin-based hydrogel being a supersaturated solution, i.e. 100% antibiotic in deionized water. The invention also provides a method for preparing the hydrogel and antibiotic composition based on the silk fibroin. The method comprises the following steps: preparing a 5% silk fibroin solution and keeping the temperature at 16-21 DEG C; adding a saturated antibiotic solution to the silk fibroin solution to form a silk fibroin-based hydrogel; stabilizing the hydrogel at 16 to 21 DEG C; and lyophilizing the stable hydrogel at 4-6 DEG C to form a dry hydrogel silk fibroin matrix having a drug embedded therein. In addition, the present invention provides compositions of drug-silk fibroin-based hydrogel formulations and methods for their use in wound healing of extraction fossa.

Description

Technical Field

[0001] The present invention relates to a sustained drug delivery method for wound healing using silk hydrogel and its products. More specifically, a method for preparing a silk hydrogel preparation that can release antibacterial agents and osteoinductive agents at a sustained and controllable rate to promote wound healing in tooth extraction sockets. Background Art

[0002] Dental procedures leading to tooth extraction include acute / chronic pulpitis, apical periodontitis / abscesses, severe periodontitis, and alveolar bone loss leading to tooth movement. The associated bacterial infection is of a particularly localized nature, confined to the pulp tissue and periapical region within the alveolar bone. Once the source of infection, including the decayed tooth or root, is removed, the wound will heal without secondary complications in a healthy person. The use of systemic antibiotics after tooth extraction is the most common practice among dentists worldwide. The most common reason for this practice is to prevent secondary infection of the post-extraction wound.

[0003] Generally, antibiotics are not required if the infection is localized and there is no abscess or pus formation, which is common in conservative tooth extraction cases. However, considering the general clinical scenario, the limited strict sterilization protocols in the clinic, the poor oral hygiene practices of the population (patients), dentists prefer oral systemic antibiotics, including amoxicillin, amoxicillin + clavulanic acid, fluoroquinolones, tetracycline / doxycycline, etc. It is also believed that the use of such systemic antibiotics can cause side effects such as gastric irritation, nausea, vomiting, and diarrhea, making the patients uncomfortable. In addition, overuse of antibiotics can lead to antibiotic resistance.

[0004] In this case, dentists will typically use any surgical dressing, which requires surgical retrieval and has no antibiotic activity of its own. In addition, it is worth noting that there are currently no surgical dressings or gages specifically designed for tooth extraction or wound management. Therefore, there remains a need for a composition composed of a natural polymer medium that has a medically relevant, biocompatible, and mechanically feasible structure to deliver drugs to the target delivery area to effectively prevent or treat infection.

[0005] Existing technology:

[0006] CN110090320A provides a conical structure for restoring a tooth extraction socket with antibacterial activity. The conical structure is solidified by the repair material to be formed, and a layer of collagen dense membrane is adhered to the bottom of the conical structure. The repair material described includes collagen-hydroxyapatite composite material, collagen, and minocycline hydrochloride, wherein collagen-hydroxyapatite is a multiple condensation material, and the mass ratio of collagen to broad-spectrum antibiotics is 1-6:0-6:0.1-1.5, and collagen is not 0. The collagen dense membrane is squeezed and collected after the collagen solution is freeze-dried. The method comprises: vacuum freezing the collagen solution to obtain a collagen sponge, densifying the collagen sponge to obtain a collagen membrane; preparing a collagen-hydroxyapatite suspension with collagen, calcium hydroxide and phosphoric acid, adding the collagen solution and a material containing a broad-spectrum antibiotic to obtain a mixed solution; injecting the mixed solution into a mold, filling the mold with the mixed solution, covering the mold opening with a dense collagen membrane, and performing vacuum freeze drying. The mold should accommodate a tool with a consistent tooth extraction socket shape.

[0007] CN111849013A provides a method for preparing a nano-hydroxyapatite-silk fibroin mineralized scaffold. The silk fibroin scaffold is immersed in a mineralizing solution for reaction to obtain a silk fibroin scaffold; the mineralizing solution is a mixed aqueous solution of ethylenediaminetetraacetic acid (concentration 0.2-0.3 mol / L) and sodium calcium and sodium dihydrogen phosphate (concentration 0.1-0.2 mol / L). The silk fibroin scaffold is immersed in the mineralizing solution and reacted for 12-24 hours under reaction conditions of 120-130°C and 1-3 atmospheres. Preferably, the reaction conditions are 121°C and 2 atmospheres for 24 hours. In addition, it also includes freeze drying at -80°C for 12 hours and then freeze drying at -50°C. This requires expensive chemicals and maintaining very high temperatures and freezing temperatures for at least 24 hours. This significantly increases production costs.

[0008] The use of silk fibroin and its use in wound healing has been well documented. However, there is still much room for optimization of existing technologies to provide better drug retention and sustained drug delivery to promote wound healing in extraction sockets. In addition, the oral cavity has a very dynamic environment, so any sustained and uninterrupted application of formulations is very difficult.

[0009] Purpose of the Invention

[0010] Therefore, in order to overcome the shortcomings of the prior art, the main object of the present invention is to provide a silk fibroin-based hydrogel preparation, which can continuously and controllably release drugs, more especially antibacterial agents and osteoinductive agents, at a sustained and controllable rate, and more especially formulate the preparation for wound healing of tooth extraction sockets.

[0011] Another object of the present invention is to provide an economical solution for local delivery of antibiotic drugs at the extraction site which can be easily placed at the extraction site by the dentist / medical practitioner.

[0012] Another object of the present invention is to provide a silk fibroin-based hydrogel preparation that can effectively and continuously release antibiotic drugs for 3-5 days to prevent secondary infection at the tooth extraction wound site, thereby reducing the use of systemic antibiotics.

[0013] Another object of the present invention is to provide a silk-based hydrogel formulation that can form a suitable microenvironment to control bacterial secondary infection and enhance alveolar bone regeneration. Summary of the invention

[0014] In achieving the above-mentioned object of the present invention, the present invention provides a sustained drug delivery method for wound healing using silk hydrogel and a product for wound healing thereof. More specifically, the present invention provides a hydrogel preparation based on silk fibroin, which can release antibacterial agents and osteoinductive agents at a sustained and controllable rate.

[0015] In the main embodiment of the present invention, the present invention provides a silk fibroin-based hydrogel and antibiotic composition for wound healing of tooth extraction sockets, wherein the silk fibroin-based hydrogel and antibiotic composition comprises 5% silk fibroin embedded with 0.4-20% antibiotic, more specifically, tetracycline or doxycycline, and the antibiotic used to prepare the silk fibroin-based hydrogel is a supersaturated solution, more specifically, 100% concentration (100 mg / ml) of antibiotic. The hydrogelation process is achieved by adding 100% concentration (100 mg / ml) of antibiotic to 5% silk fibroin.

[0016] Silk fibroin-based hydrogels enable drug delivery and are suitable for controlled release of antimicrobial agents while promoting regeneration of injured cells. The hydrogels contain freeze-dried doxycycline or tetracycline on a silk fibroin support structure. Freeze-dried gels remove water molecules and extend shelf life.

[0017] In another embodiment of the present invention, the present invention provides a method for preparing a silk-based hydrogel formulation for wound healing of tooth extraction sockets, the method comprising the following steps:

[0018] a) preparing a 5% silk fibroin solution;

[0019] b) taking 500 μl of the prepared 5% silk fibroin solution and keeping it at 16-21° C.;

[0020] c) preparing a supersaturated solution of tetracycline / doxycycline drug by adding 100 mg of drug to 1 ml of deionized water and vortexing for 30-60 seconds to prepare a 100% concentration drug solution;

[0021] d) adding a saturated tetracycline / doxycycline solution to the 5% silk fibroin solution to form a silk fibroin-based hydrogel;

[0022] e) stabilizing the prepared hydrogel at a temperature of 16-21° C. for the first 8 hours;

[0023] f) then refrigerated at 4-6°C for the next 3 days (72 hours); and

[0024] f) freeze-drying the stabilized prepared hydrogel to form a dry hydrogel silk fibroin matrix having the drug embedded therein.

[0025] The prepared silk-based drug hydrogels showed a large amount of drug release in the first 3-5 days and then a small amount of drug release in the next 15 days.

[0026] In another embodiment of the present invention, there is provided a method for using a drug-silk fibroin-based hydrogel formulation for wound healing of a tooth extraction socket, the method comprising:

[0027] Cleaning the socket after tooth extraction;

[0028] The freeze-dried silk drug hydrogels were standardized using deionized water to introduce flexibility;

[0029] remodeling of standardized freeze-dried silk drug hydrogels to resemble the contours of extracted tooth roots;

[0030] Placing a standardized freeze-dried silk drug hydrogel in the extraction socket after tooth extraction; and

[0031] A standardized freeze-dried silk drug hydrogel was retained in a tooth socket embedded in a blood clot to enhance wound healing and osteoinduction;

[0032] in,

[0033] Silk-based drug hydrogels release drugs (tetracycline / doxycycline) locally in a controlled manner at microbiocidal concentrations (local concentrations must be optimized: MIC of 1.2 ul / ml body fluid) within the first 4-5 days; and

[0034] The silk-based drug hydrogel released the drug locally over the next 15 days at levels below the microbial killing concentration.

[0035] In another embodiment of the present invention, a novel silk-based hydrogel system is provided, wherein its biodegradation is enhanced by using proteinase-K. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The purpose of the present invention may be understood in more detail, and the description of the present invention briefly outlined above may be understood more specifically, by reference to certain embodiments of the present invention illustrated in the accompanying drawings, which form a part of this specification. It should be noted, however, that the accompanying drawings illustrate preferred embodiments of the present invention and are therefore not to be considered as limiting the scope thereof, as the present invention may admit of other equally effective equivalent embodiments.

[0037] Figure 1 is a schematic diagram of using a silk-based hydrogel formulation for post-tooth extraction wound management according to an embodiment of the present invention;

[0038] Figure 2 is the optical absorption graph of SF hydrogel with doxycycline at 550 nm;

[0039] Figure 3 Scanning electron micrographs depicting topological analysis of hydrogel samples (A) silk hydrogel without doxycycline, (B) doxycycline, and (C) SF hydrogel with doxycycline are depicted, Scale: 100 μm;

[0040] Figure 4 A and 4B provide FTIR spectra of silk fibers alone, doxycycline alone, and lyophilized samples of SF and doxycycline hydrogel;

[0041] Figure 5 Depicted are photographs of inhibition zone studies of S. aureus with SF (A), doxycycline (B), and SF-doxycycline (C) and photographs of inhibition zone studies of S. mutans with SF (D), doxycycline (E), and SF-doxycycline (F);

[0042] Figure 6 A graphical representation of cell viability in MC3T3, HDFC, MG63 cell lines by MTT assay in the presence of negative control, SF hydrogel, doxycycline alone, and SF-doxycycline hydrogel is provided;

[0043] Figure 7 Graphical representations of drug release profiles of silk alone, doxycycline alone, and SF-doxycycline hydrogels are provided;

[0044] FIG8 provides a) a rabbit mouth image indicating the location of the incisor extraction and the placement of the SF-doxycycline hydrogel, b) a rabbit mouth image indicating the postoperative suture, and c) a rabbit mouth image showing wound healing on day 7; and

[0045] FIG. 9 depicts pathological images of injured tissues of rabbit groups on day 14 after follow-up treatment, a) untreated or control group, b) SF-doxycycline hydrogel group, c) systemic antibiotic treatment group, and d) vehicle control or SF hydrogel group. DETAILED DESCRIPTION

[0046] The present invention will be described more fully below with reference to the accompanying drawings, in which preferred embodiments of the present invention are shown. However, the present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, the embodiments are provided to make the present disclosure more comprehensive and to fully convey the scope of the present invention to those skilled in the art.

[0047] Silk is a high-quality natural fiber obtained from silkworms, mainly composed of silk fibroin (SF) accounting for about 75%. Silk fibroin hydrogels are considered to be effective candidates for many applications, especially in the biomedical field, due to their unique biocompatibility and biodegradability. Silk fibroin is usually compounded with natural or synthetic polymers to prepare hydrogels. In addition, silk fibroin hydrogels can be prepared by physical crosslinking and chemical crosslinking. It is known that the self-assembly of SF into hydrogels is induced at 37°C. In addition, a mixed solution of silk fibroin and sodium alginate (SA) that can be left to stand at 37°C causes SF and SA molecules to self-assemble to form a hydrogel. In addition, methods such as ultrasonic treatment, shearing, electric field effect, pH adjustment, etc. are known to induce physical crosslinking to form a hydrogel. Chemical treatments are known to induce hydrogel formation, for example, photopolymerization, high-intensity γ-ray irradiation, and the use of reagents, such as genipin, glutaraldehyde, carbodiimide, and enzymes. All of these methods involve the use of high temperatures, expensive reagents, and procedures.

[0048] The present invention provides an extremely simple method to induce the hydrogel formation of SF embedded with antibiotics.

[0049] In one embodiment of the present invention, a silk-based hydrogel formulation for post-extraction wound management and methods of making and using the same are provided. The present invention provides a novel silk-based hydrogel system for drug delivery suitable for sustained release of drug molecules with antimicrobial properties and regenerative potential. In one embodiment, hydrogels were developed with silk fibroin by adding doxycycline / tetracycline and forming a "drug-induced" hydrogel, which was freeze-dried to remove water molecules and extend shelf life.

[0050] In another embodiment of the present invention, a novel silk-based hydrogel is provided, which can release antibacterial agents and osteoinductive agents at a sustained and controllable rate. Preferably, the prepared silk-based drug hydrogel releases a large amount of drug in the first 3-5 days and shows a small amount of drug release in the next 15 days.

[0051] In another embodiment of the present invention, the present invention provides a silk fibroin-based hydrogel and antibiotic composition for extraction socket wound healing, wherein the silk fibroin-based hydrogel and antibiotic composition comprises 5% silk fibroin embedded with 0.4-20% antibiotic, more specifically, tetracycline or doxycycline, and the antibiotic used to prepare the silk fibroin-based hydrogel is a supersaturated solution, more specifically, a 100% concentration (100 mg / ml) of antibiotic.

[0052] In another embodiment of the present invention, in order to prepare a novel hydrogel based on silk, 5% silk fibroin solution and 500ul solution are taken in (16-21 degrees Celsius). A supersaturated solution of tetracycline / doxycycline (100mg / ml) is prepared and vortexed for 30-60 seconds (immediately after adding 100mg of the drug in 1ml of DI water). Supersaturated tetracycline / doxycycline solution is added to the 5% silk fibroin solution to form a hydrogel immediately. The hydrogel is then stabilized for 8 hours at room temperature (16-21 degrees Celsius) and kept for 3 days (72 hours) at 4-6 degrees Celsius. After refrigeration, the gel is freeze-dried, and the freeze-dried gel will be a dry hydrogel silk matrix with drugs embedded therein. The freeze-dried hydrogel will show a release curve, wherein a large amount of drug release lasts for 3-5 days, and then a small amount of drug release lasts for 15 days.

[0053] Table 1 shows that different ratios of silk solution and tested drug solution have been established for forming silk-based hydrogels, as follows:

[0054] Table 1

[0055]

[0056]

[0057] refer to Figure 1 , which is a schematic diagram of using a silk-based hydrogel formulation for post-tooth extraction wound management according to an embodiment of the present invention. The lyophilized silk drug hydrogel can be placed in the tooth extraction socket after tooth extraction, wherein the hydrogel will remain in the socket and be embedded in the blood clot. Based on the hydrogel, the drug (tetracycline / doxycycline) is released locally in a controlled manner through a microbial killing concentration (local concentration must be optimized: MIC is 1.2ul / ml body fluid) within the first 4-5 days and below the microbial killing concentration for the next 15 days, which will lead to host regulatory activity (anti-collagenase activity of doxycycline / tetracycline) and enhance wound healing and bone induction.

[0058] Example

[0059] Example 1

[0060] Silk purification

[0061] To extract silk fibroin, cut the cocoons into small pieces and remove the silkworms. Take 2 grams of cocoon pieces. Take two 500 ml beakers, take 200 ml of deionized water, cover with aluminum foil and heat to boiling. Take 0.424 grams of sodium carbonate and add to two glass beakers respectively, add boiling water (to prepare 0.02M Na2CO3 solution), add the cut cocoons (2g) to one of the beakers and keep it under constant stirring (200rpm) for 15 minutes. After 15 minutes, remove the silk fibroin and add boiling DI water (Na2CO3) again in the other beaker. 2 CO 3 0.02M Na solution) and kept for 15 minutes in the same way as before. After boiling for a total of 30 minutes (15+15), the silk fibroin was removed with a scraper and rinsed in cold water 4-5 times to remove residual Na 2 CO 3 . Discard the remaining solution. Squeeze the silk fibroin thoroughly to remove moisture, spread in clean aluminum foil, and keep in a hot air oven at 60°C overnight to dry. Store it in clean aluminum foil. Extract about 0.65-0.7 grams of silk fibroin by degumming 1 gram of silk fiber. Prepare a 9.3M LiBr solution to make a 20% (wt / vol) solution. (For every 1 gram of silk fibroin, prepare 10 ml of 9.3 molar LiBr solution by adding 8.07 grams of LiBr and make it up to 10 ml).

[0062] Silk fibroin (SF) fibers were placed in a round-bottom flask and placed in a hot water bath at 60°C. Freshly prepared LiBr solution was made and immediately poured into the round-bottom flask so that the entire SF fiber was immersed therein and kept under constant stirring at 200rpm for 4 hours. Once SF was completely dissolved, a transparent amber solution was formed. The transparent amber SF fiber solution was collected, placed in a dialysis membrane and bundled so that it would not leak, and stored in a 1L flask filled with DI water for dialysis. The water was changed after 1h, 4h, 12h, 24h, 36h, 48h, 60h and 72h respectively. After the dialysis was completed, the solution was taken out and extracted in a 50ml centrifuge tube. The solution was then centrifuged twice at 9000rpm for 20 minutes to remove visible impurities. The centrifuged samples (silk fibroin solution) were then collected and stored at 4°C.

[0063] Three sets of 100ul solutions were placed in pre-weighed small plastic boats and kept dry in a hot air oven at 60°C overnight. The dry weight of the silk was calculated and divided by 100ul to obtain the w / v% concentration of the silk. Typically, a 5% (w / v) silk solution was obtained.

[0064] Example 2

[0065] Hydrogel preparation

[0066] Silk doxycycline hydrogels were prepared using a precise combination of silk fibroin and supersaturated doxycycline solution at a specific temperature and in a timed process.

[0067] Place 500ul of 5% silk fibroin solution in a 2ml pipette (16-24°C), and add 100-20ul of freshly prepared supersaturated doxycycline hydrochloride (1ml of 100mg / ml doxycycline is prepared within 1 minute and vortexed thoroughly, and added to the prepared silk fibroin solution).

[0068] The regenerated silk fibroin-doxycycline hydrogels started to form and were placed at room temperature for 8 h, then kept at 4 °C for 3 days, and then frozen at -80 °C. The hydrogels were then freeze-dried.

[0069] Table 2

[0070]

[0071]

[0072] After the addition of doxycycline solution, SF formed a gel-like structure immediately. The formed hydrogel was formed without stirring. The initial matrix of the hydrogel formulation occurred immediately and the hydrogel matrix formulation was stable after 8 hours as found in the optical stability study.

[0073] Example 3

[0074] Gelation kinetics

[0075] SF-doxycycline hydrogels were prepared in 96-well plates with different final concentrations of doxycycline (10-, 20-, 30-, 40-, and 50-mg / ml). OD measurements were performed at 550 nm in a multiplate reader (Tecan-Infinite M-200Pro).

[0076] The dynamics of the hydrogels were determined by measuring the absorbance at 550 nm. Figure 2 As shown, after 8 hours, the absorbance of all SF-doxycycline hydrogels increased, and the absorbance was the highest in the SF hydrogel containing 50 mg of doxycycline. The SF hydrogel containing 40 mg of doxycycline showed the smallest absorbance. The SF hydrogel without doxycycline showed no absorbance. The absorbance was stable (flat in the figure) within 8-20 hours, indicating that there was no matrix formation or changes indicating that the SF-doxycycline hydrogel was stable, and the SF-doxycycline hydrogel matrix formation was stable for 8-12 hours.

[0077] Example 4

[0078] a) Water absorption and swelling study

[0079] The freeze-dried hydrogel (W o ) were weighed and immersed in double distilled water at 37 °C at different time intervals, from 0 h to 90 h. t ) were weighed, and the swelling ratio (SR) was calculated by Equation 1.

[0080] Equation 1: Swelling Ratio (SR) Calculation

[0081]

[0082] Among them, W t is the mass of swelling obtained at time t, W o is the mass of the xerogel obtained at time 09.

[0083] The net weight and swelling ratio of the hydrogel increased for up to 4 hours until the hydrogel reached equilibrium. After 4 hours, the net weight and swelling ratio began to decrease (drug was washed off while continuing to wet). No dimensional changes in the hydrogel were observed for up to 12 hours, as shown in Table 3. The dry weight was 25 mg.

[0084] Table 3: Water absorption and swelling properties of SF doxycycline hydrogel

[0085]

[0086]

[0087] b) Absorbance of SF hydrogel containing doxycycline:

[0088] The dynamics of the hydrogels were determined by measuring the absorbance at 550 nm. Figure 2 As shown, after 8 h, the absorbance of all SF-doxycycline hydrogels increased, with the highest absorbance in the SF hydrogel containing 50 mg doxycycline. The SF hydrogel containing 40 mg doxycycline showed the smallest absorbance. The SF hydrogel without doxycycline showed no absorbance. The SF-doxycycline hydrogel matrix formed was stable for 8-12 hours.

[0089] Example 5

[0090] Topological analysis by scanning electron microscopy

[0091] The freeze-dried samples were fixed to the scanning electron microscope (SEM) sample holder using carbon tape. The hydrogel and doxycycline were coated with gold-palladium (Au-Pd; 80:20) using a sputtering coater. The samples were examined in a HITACHI (S3700N) scanning electron microscope (SEM) at an accelerating voltage of 20 kV using a SE detector. Micrographs were taken at a magnification of 500 times.

[0092] like Figure 3 As shown in the SEM image of A, SF hydrogel has a porous leaf-like structure. Figure 3 As shown in the SEM image of B, doxycycline showed irregular cylindrical and cubic morphology. Figure 3 As shown in the SEM image of C, SF-doxycycline hydrogel exhibits fibrous and porous structures.

[0093] Example 6

[0094] Fourier Transform Infrared (FTIR) Spectroscopy

[0095] Lyophilized samples were analyzed by FTIR spectroscopy. Spectra were smoothed with a constant smoothing factor for comparison. Figure 4 FTIR spectra of silk fibers alone, doxycycline alone, and lyophilized samples of SF and doxycycline hydrogel are provided.

[0096] Silk:

[0097] The FT-IR spectrum of the silk protein standard showed that the characteristic vibration band near 3279.88 cm-1 showing NH and OH stretching 1620 cm-1 was assigned to the absorption peak of the amide I peptide backbone (C=O stretching), the band near 1519 cm-1 was assigned to amide II (NH bending), and the band near 1236.91 cm-1 was assigned to amide III (CN stretching).

[0098] Doxycycline:

[0099] The FT-IR spectrum of the doxycycline standard showed that the characteristic vibration band near 3279.19 cm-1 showing NH and OH stretching 1662.28 cm-1 was assigned to the absorption peak of the amide I peptide backbone (C=O stretching), the band near 1552.73 cm-1 was assigned to amide II (NH bending), and the band near 1216.74 cm-1 was assigned to amide III (CN stretching).

[0100] Silk and Doxycycline Hydrogel:

[0101] The combination of silk and doxycycline hydrogels showed similar peaks compared to the control silk fibroin and doxycycline.

[0102] FT-IR spectra of silk and doxycycline hydrogels showed absorption peaks of the peptide backbone near 3279.87 cm-1 and characteristic vibration bands showing NH and OH stretching at 1620.30 cm-1 were assigned to amide I (=O stretching), the band near 1521.92 cm-1 was assigned to amide II (NH bending), and the band near 1235.84 cm-1 was assigned to amide III (CN stretching).

[0103] Example 7

[0104] X-ray diffraction

[0105] The diffraction patterns of the freeze-dried samples were measured using an X-ray diffractometer XRD (platinum ATR-αII) operating at 40 kV, 30 mA and a scanning speed of 2° per minute. The diffraction indices were recorded at 10-80°. Figure not provided.

[0106] Example 8

[0107] Biological Research

[0108] a) Inhibition zone

[0109] Staphylococcus aureus and Staphylococcus variants were cultured in nutrient broth and plated on agar plates. Hydrogel (25 mg of SF doxycycline hydrogel with a diameter of 5 mm, with 2 mg of drug) was kept in each plate in the center on the medium, and for doxycycline, the drug was added in the center of the flushed medium. The plates were incubated at 37°C for 24 hours. After the incubation period, the radius of the inhibition zone was measured in each plate.

[0110] like Figure 5 As shown in A and 5D, S. aureus and S. mutans did not show inhibition zones for SF alone. Figure 5 As shown in B and E, the inhibition zones were 10 mm and 25 mm for Staphylococcus aureus and Staphylococcus mutans, respectively, when doxycycline was used. Figure 5 As shown in C and 5F, the inhibition zone was smaller with SF-doxycycline compared to doxycycline alone. In S. aureus, the inhibition zone was 7 mm, while in S. variabilis, the inhibition zone was 21 mm, indicating active doxycycline and restricted release of doxycycline in the SF-doxycycline hydrogel. This clearly shows the controlled release of the drug from the SF hydrogel.

[0111] b) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay

[0112] All three cell lines (10,000 cells / 200 μl culture medium) were cultured in their respective culture medium in 96-well plates until 60% confluence was reached. Hydrogel and doxycycline were added to their respective wells. The plates were incubated at 37°C with 5% CO 2 Incubate for 24 hours. Remove the culture medium and add 200 μl of MTT solution (0.5 mg / ml, prepared in phosphate buffered saline (PBS, Himedia, catalog number TS1119)) to each well. Incubate the plate in the dark at 37°C with 5% CO 2Incubate for 4 hours. Then remove the MTT solution and add 100 μl of demethyl sulfoxide (DMSO) to each well to dissolve the formazan crystals. Calculate the absorbance at 570 nm in a multiplate reader. Calculate the cell viability percentage using equation 2.

[0113] Equation 2 Calculation of cell viability percentage

[0114]

[0115] Where OD is the optical density at 570 nm.

[0116] like Figure 6 As shown in Figure 2, all three cell lines MC3T3, HDFC, and MG63 (3 different cell lines were studied) showed the lowest viability (40%) under doxycycline plain. However, SF-doxycycline hydrogel showed about 75%-80% cell viability compared to the control, which was similar to the cell viability of silk fibroin solution. This clearly shows that SF-doxycycline hydrogel is biocompatible compared to the drug alone.

[0117] In a study of gingival fibroblasts, the cell viability of the SF-doxycycline hydrogel was 87%.

[0118] c) Release curve

[0119] The sample (25 mg of SF doxycycline hydrogel containing 2 mg of drug) was placed in a pipette and 1 ml of 0.9% saline (artificial saliva) was added to each tube. The tube was kept in an incubator (REMI, catalog number RHI80) at 37°C. 100 μl samples were taken from each tube at different time intervals to measure the OD value at 275 nm using an ultraviolet spectrum (Shimadzu UV-2600). The OD value was measured for up to 168 hours and plotted in a chart.

[0120] The release profile of SF-doxycycline hydrogels was studied in phosphate buffer solution. Figure 7As shown in , after an initial burst of 20% drug release in the first hour, 70% of the drug is continuously released over the next 24 hours. Subsequently, the drug is released in a very slow and sustained manner over the next 15 days. To hint at the real situation in dental wounds, when the hydrogel is inserted into the extraction socket, the release of doxycycline will be limited due to the semi-solid nature of the blood clot surrounding the hydrogel in the wound. Therefore, the drug will be released over a longer time frame. 20% of the initial drug release will be released within the first 3-4 hours, and the remaining 70% of the drug will be released within the next 48-72 hours (3 days based on conventional systemic antibiotic / microbial concentrations in local wounds), followed by subsequent submicrobial concentrations over the next 15 days for host regulation and anti-collagenase activity (enhanced wound healing).

[0121] d) Animal research

[0122] Animal studies were conducted in New Zealand rabbits in which incisor teeth were extracted and SF-doxycycline hydrogel was placed at the wound site.

[0123] like Figure 8a As shown in , an incisor was extracted in a rabbit mouth, a wound was created, and SF-doxycycline hydrogel was placed at the wound site. Figure 8b Postoperative suturing is described. Figure 8c After 7 days as shown, the wound was completely healed. This shows that the SF-doxycycline hydrogel is effective in wound healing.

[0124] For further analysis, four groups of rabbits were used, in which the incisors were extracted, wounds were created, and the following treatments were performed: a) control - no antibiotics, b) SF-doxycycline hydrogel group, c) antibiotic group - systemic antibiotics (4-5 mg doxycycline / day), and d) vehicle control - SF hydrogel. On the 14th day of treatment, the alveolar socket tissue, i.e., tissue from the wound site, was removed from all four groups and subjected to histopathological analysis.

[0125] Figure 9a Histopathological images of untreated or control groups are depicted, showing incomplete epithelial coverage on the superficial surface with chronic inflammatory cell infiltration and sprouting endothelial cell proliferation in the lamina propria.

[0126] Fig.9c Histopathological images of the systemic antibiotic treatment group are depicted, showing irregular epithelial stratification with variable thickness. The underlying lamina propria showed a mild chronic inflammatory cell infiltration.

[0127] Figure 9d Histopathological images of the vehicle control or SF hydrogel groups are depicted, showing an irregular outer epidermal surface. The lamina propria showed a dense connective tissue stroma with mild inflammatory cell infiltration and hemorrhagic areas.

[0128] However, Figure 9b The histopathological images of the SF-doxycycline hydrogel group are depicted, showing a smooth and uniform outer epidermal surface. The lamina propria showed a dense connective tissue stroma with mild inflammatory cell infiltration. This clearly showed that the SF-doxycycline hydrogel completely healed the wound compared to all other 3 groups.

[0129] In addition, gene expression studies showed that the wound healing results of the SF-doxycycline hydrogel group were positive. The gene expression of bFGF, VEGF, and TGF in the SF-doxycycline hydrogel group was higher than that in the systemic antibiotic group.

[0130] Although certain exemplary embodiments have been described and shown in the accompanying drawings, it should be understood that such embodiments are merely illustrative and not limiting of the broad invention, and the invention is not limited to the specific configurations and arrangements shown and described, as various other changes, combinations, omissions, modifications and substitutions are possible in addition to those set forth in the above paragraphs. Those skilled in the art will appreciate that various adjustments and modifications of the just described embodiments may be configured without departing from the scope and spirit of the invention.

Claims

1. A silk fibroin-based hydrogel and antibiotic composition for wound healing in tooth extraction sockets, in, The silk fibroin-based hydrogel and antibiotic composition comprises 5% silk fibroin embedded with 0.4-2% antibiotic, more specifically, tetracycline or doxycycline, The antibiotic used to prepare the silk fibroin-based hydrogel is a supersaturated solution, i.e., a 100% (100 mg / ml) concentration of the antibiotic in deionized water, and The silk fibroin-based hydrogel and antibiotic composition was formed by adding 100% (100 mg / ml) concentration of antibiotics to 5% silk fibroin to form a hydrogel.

2. The silk fibroin-based hydrogel and antibiotic composition according to claim 1, wherein: The composition was able to achieve a large amount of drug release within the first 3-5 days, and then showed a small amount of drug release within the next 15 days.

3. The silk fibroin-based hydrogel and antibiotic composition according to claim 1, wherein: The composition is capable of achieving local release of the antibiotic in a controlled manner with a microbial killing concentration of MIC 1.2 ul / ml body fluid within the first 4-5 days.

4. The silk fibroin-based hydrogel and antibiotic composition according to claim 1, wherein: The composition includes the antibiotic tetracycline or doxycycline.

5. A method for preparing a silk fibroin-based hydrogel and an antibiotic composition for wound healing of tooth extraction sockets, the method comprising the following steps: a) preparing a 5% silk fibroin solution and maintaining it at 16-21° C.; b) preparing a supersaturated antibiotic solution in deionized water and vortexing for 2-3 minutes to prepare a 100% antibiotic solution; c) adding a saturated antibiotic solution to the 5% silk fibroin solution to form a silk fibroin-antibiotic based hydrogel; d) stabilizing the prepared hydrogel at a temperature of 16-21° C. for the first 8 hours; and refrigerating at 4-6° C. for the next 72 hours, e) freeze-drying the stabilized prepared hydrogel to form a dry silk fibroin-based hydrogel matrix having the antibiotic embedded therein, in, The silk fibroin-based hydrogel and antibiotic composition comprises 5% silk fibroin embedded with 0.4-2% antibiotic, more specifically, tetracycline or doxycycline, and The antibiotic used to prepare the silk fibroin-based hydrogel was a supersaturated solution, ie, 100% concentration of antibiotic in deionized water.

6. The method according to claim 5, wherein: The method provides a silk fibroin-based hydrogel and antibiotic composition that is capable of achieving a large amount of drug release within the first 3-5 days and then exhibits a small amount of drug release within the next 15 days.

7. The method according to claim 5, wherein: The method provides a silk fibroin-based hydrogel and antibiotic composition capable of achieving local release of the antibiotic in a controlled manner with a microbial killing concentration of MIC 1.2 ul / ml body fluid within the first 4-5 days.

8. The method according to claim 5, wherein: The method provides a silk fibroin-based hydrogel and an antibiotic composition, including the antibiotic tetracycline or doxycycline.

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