A sPL nanomaterial for repairing gingival recession and its preparation method

Abstract

This invention relates to an sPL nanomaterial for repairing gingival recession and its preparation method, specifically to an sPL nanomaterial for repairing gingival recession and its preparation method. The nanomaterial is composed of platelet lysis buffer, exosomes, graphene oxide, and CREKA peptide, wherein the exosomes are extracted from dental pulp stem cells. The method includes: Step 1: Extraction of exosomes; Step 2: Preparation of sPL; Step 3: Preparation of the Go-sPL-CREKA nanomaterial. The nanomaterial of this invention can promote the proliferation of gingival fibroblasts, increase the content of growth factors related to gingival recession repair, and enhance the repair effect of gingival recession. This invention is used for repairing gingival recession.

Description

Technical Field

[0001] This invention relates to the field of materials for repairing gingival recession, and more particularly to an sPL nanomaterial for repairing gingival recession and its preparation method. Background Technology

[0002] Periodontal disease is one of the most common diseases worldwide. Besides periodontitis and gingivitis, other diseases and changes can also affect periodontal tissues. However, periodontitis has the greatest impact on adult oral health and public health. Periodontitis is a chronic, inflammatory, non-contagious disease that affects all parts of the periodontal tissues and causes irreversible damage. The prevalence of this disease gradually increases with age. With the increasingly prominent aging population in my country, periodontitis will become an even more prominent problem.

[0003] Gingival recession (GR) is a common symptom of periodontitis, referring to the recession of the gingival margin at the cementoenamel junction towards the root, sometimes accompanied by the receding of the interdental papillae, resulting in root exposure and affecting aesthetics. Severe gingival recession may involve some alveolar bone resorption and attachment loss. In recent years, the restorative and aesthetic problems caused by gingival recession have received considerable attention. Gingival recession and periodontitis influence each other; gingival recession can trigger periodontitis, and periodontitis can directly lead to gingival recession. If gingival recession is accompanied by periodontitis, it will exacerbate the recession and accelerate alveolar bone resorption. This vicious cycle leads to a decrease in gingival attachment, resulting in widening gaps between teeth, root exposure, and tooth loosening. Therefore, it requires sufficient attention. Because severe gingival recession leads to root exposure, self-restoration alone is often insufficient, and surgical restoration is usually required clinically. However, this method has limitations and is relatively invasive.

[0004] In recent years, stem cell-based tissue engineering technology has become a key focus and hot topic in the field of dentistry. Currently, the commonly used method for repairing gingival recession is to add growth factors to the material. However, adding a single growth factor has a limited effect, a short half-life, and poor prevention and repair effects; adding multiple growth factors requires advanced technology and is difficult to operate. Summary of the Invention

[0005] The present invention aims to address the problem that existing methods have limited growth factor activity and poor prevention and repair effects, and provides an sPL nanomaterial for repairing gingival recession and its preparation method.

[0006] The sPL nanomaterial of this invention for repairing gingival recession is made of platelet lysate, exosomes, graphene oxide and CREKA peptide, wherein the exosomes are extracted from dental pulp stem cells.

[0007] The present invention provides a method for preparing sPL nanomaterials for repairing gingival recession, comprising the following steps:

[0008] Step 1: Extraction of exosomes

[0009] The purified rat dental pulp stem cells were cultured in complete culture medium to the third generation. When the cells adhered and grew to 50%-60%, the culture medium was replaced with the appropriate exosome-free medium and cultured for a longer period. When the cells adhered and grew to 90%-100%, the culture supernatant was collected and the exosomes were extracted.

[0010] Step 2: Preparation of SPL

[0011] Platelet lysis buffer PL was prepared, and PL was mixed with exosomes to obtain sPL;

[0012] Step 3: Preparation of the nanomaterial Go-sPL-CREKA

[0013] Graphene oxide was mixed with the sPL prepared in step two to obtain a Go-sPL suspension. Then, CREKA peptide was added and stirred for 2-3 hours to prepare the nanomaterial Go-sPL-CREKA.

[0014] Furthermore, the specific method for preparing the platelet lysis buffer PL in step two is as follows:

[0015] Platelet enrichment was performed by adding CaCl2 to the enriched platelets, freezing them at -80℃ for 8-10 hours, and then thawing them at 37℃ for 5-10 minutes. This freeze-thaw cycle was repeated 10 times. The lysate from the last thaw was centrifuged at 6500 rpm for 15-20 minutes, and the supernatant was retained as platelet lysate (PL).

[0016] The platelet enrichment method is specifically as follows:

[0017] Centrifuge whole blood at 1500-2700 rpm for 15-20 min to collect the upper plasma layer, which is platelet-rich plasma. Centrifuge the platelet-rich plasma again at 3500-4300 rpm for 20-25 min and collect the lower layer of platelets. The volume of the platelets enriched is 1 / 10 of the whole blood volume.

[0018] Furthermore, in step two, the volume ratio of PL to exosomes is (40-60):1.

[0019] Furthermore, in step three, the concentration of graphene oxide in the Go-sPL suspension is 25-50 μg / mL.

[0020] Furthermore, in step three, the volume ratio of Go-sPL suspension to CREKA peptide is 1:(2-5).

[0021] This invention encapsulates specifically activated sPL (splastic protease) using graphene oxide (Go) as a nanocarrier and modifies it with the peptide CREKA to create a novel nanomaterial that promotes cell regeneration and tissue repair for the improvement and treatment of gingival recession. Focusing on promoting the proliferation and differentiation of gingival fibroblasts, this invention seeks to find a method for preparing sPL that increases the secretion of four growth factors that promote gingival fibroblast proliferation and tissue repair: VEGF, EGF, PDGF, and TGF-β. This sPL is combined with Go and modified with CREKA to directionally activate gingival fibroblasts, promoting their proliferation and self-renewal capabilities. It also targets fibrin deposited at inflamed sites, enhancing the antibacterial capacity and repair effect of gingival tissue, providing technical support and therapeutic strategies for clinical research on gingival recession using sPL nanomaterials.

[0022] The beneficial effects of this invention are:

[0023] 1. The platelet enrichment method in the invention adopts a two-step centrifugation method. The first step is to centrifuge at 1500-2700 rpm for 15 min, using low speed to separate plasma and blood cells, ensuring that platelets remain in plasma and do not settle into blood cells. All plasma from the first centrifugation is collected for a second centrifugation at 3500-4300 rpm for 20 min, so that platelets settle as much as possible at the bottom of the tube and in the bottom plasma, resulting in a lower platelet content in the upper layer and enhancing platelet concentration efficiency.

[0024] 2. The sPL in this invention is prepared by adding DPSCs exosomes to PL for further activation. It can promote fibroblast proliferation, increase the content of gingival tissue repair-related growth factors, and work synergistically with exosomes to inhibit DKK1 expression in gingival fibroblasts, activate the Wnt signaling pathway, increase the concentration of effective factors, and enhance the gingival recession repair effect.

[0025] 3. In this invention, graphene oxide itself has antibacterial and anti-inflammatory effects and can promote the expression of β-catenin, thereby activating the Wnt signaling pathway and aiding in tissue repair. As a nanocarrier encapsulating sPL, it can maintain the activity of growth factors in sPL, increase the loading of growth factors and achieve a sustained-release effect, more efficiently transporting factors to the damaged site. Simultaneously, sPL can reduce the toxicity of Go and improve biocompatibility; the two work synergistically to enhance the repair effect of gingival recession.

[0026] 4. In this invention, the CREKA peptide has the ability to specifically bind fibrin, which allows Go-sPL to target the fibrin deposited at the site of gingival inflammation and prolong its retention time at the damaged site, thereby improving the repair effect and efficiency of Go-sPL.

[0027] This invention relates to Go-sPL-CREKA nanomaterials, which utilize the rich gingival tissue repair-related growth factors and nutrients in sPL. These are encapsulated within a Go nanocarrier, and further enhanced by the addition of DPSCs exosomes to directionally activate gingival fibroblast proliferation and increase differentiation factors (such as VEGF, EGF, PDGF, and TGF-β). Combined with the targeted repair effect of CREKA peptides, this further promotes gingival fibroblast proliferation and characteristic maintenance, enhances antibacterial ability, and demonstrates good biocompatibility.

[0028] This invention can improve the treatment effect in the process of gingival recession repair and provides new ideas and methods for clinical research on periodontal diseases such as gingival recession. Attached Figure Description

[0029] Figure 1 The growth status of rat DPSCs on day 1 after passage.

[0030] Figure 2 The growth status of rat DPSCs on day 5 after passage.

[0031] Figure 3 The results of the identification of exosome surface markers;

[0032] Figure 4 To detect the toxic effects of Go on gingival fibroblasts;

[0033] Figure 5 The effect of sPL on Go cell cytotoxicity;

[0034] Figure 6 To detect the sustained release effect of Go on the cytokine VEGF in sPL;

[0035] Figure 7 To detect the sustained release effect of Go on the cytokine PDGF in sPL;

[0036] Figure 8 The proliferation of gingival fibroblasts (OD value at 450 nm);

[0037] Figure 9 The expression of DKK1 protein in gingival fibroblasts during proliferation;

[0038] Figure 10 The expression of β-atenin protein in the proliferation of gingival fibroblasts;

[0039] Figure 11 The changes in bacterial viability after treatment with graphene oxide;

[0040] Figure 12 The changes in body weight of rats in each group;

[0041] Figure 13 The expression of the COL-1 gene in gingival tissue repair;

[0042] Figure 14 The expression of the COL-2 gene in gingival tissue repair;

[0043] Figure 15 The expression of the VEGF gene in gingival tissue repair. Detailed Implementation

[0044] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0045] Specific Implementation Method 1: The sPL nanomaterial used in this implementation method for repairing gingival recession is made of platelet lysate, exosomes, graphene oxide and CREKA peptide, wherein the exosomes are extracted from dental pulp stem cells.

[0046] This embodiment encapsulates specifically activated sPL (splastic protease) using graphene oxide (Go) as a nanocarrier and modifies it with the peptide CREKA to create a novel nanomaterial that promotes cell regeneration and tissue repair for the improvement and treatment of gingival recession. Focusing on promoting the proliferation and differentiation of gingival fibroblasts, this invention seeks a method to prepare sPL that increases the secretion of four growth factors that promote gingival fibroblast proliferation and tissue repair: VEGF, EGF, PDGF, and TGF-β. This sPL is then combined with Go and modified with CREKA to directionally activate gingival fibroblasts, promoting their proliferation and self-renewal capabilities, and targeting fibrin deposited at inflamed sites, thereby enhancing the antibacterial ability and repair effect of gingival tissue.

[0047] Specific Implementation Method Two: This implementation method for preparing sPL nanomaterials for repairing gingival recession includes the following steps:

[0048] Step 1: Extraction of exosomes

[0049] The purified rat dental pulp stem cells were cultured in complete culture medium to the third generation. When the cells adhered and grew to 50%-60%, the culture medium was replaced with the appropriate exosome-free medium and cultured for a longer period. When the cells adhered and grew to 90%-100%, the culture supernatant was collected and the exosomes were extracted.

[0050] Step 2: Preparation of SPL

[0051] Platelet lysis buffer PL was prepared, and PL was mixed with exosomes to obtain sPL;

[0052] Step 3: Preparation of the nanomaterial Go-sPL-CREKA

[0053] Graphene oxide was mixed with the sPL prepared in step two to obtain a Go-sPL suspension. Then, CREKA peptide was added and stirred for 2-3 hours to prepare the nanomaterial Go-sPL-CREKA.

[0054] In step two of this embodiment, sPL is prepared by adding DPSCs exosomes to PL for further activation. It can promote fibroblast proliferation, increase the content of gingival tissue repair-related growth factors, and work synergistically with exosomes to inhibit DKK1 expression in gingival fibroblasts, activate the Wnt signaling pathway, increase the concentration of effective factors, and enhance the gingival recession repair effect.

[0055] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that the preparation method of platelet lysis buffer PL in step 2 is as follows:

[0056] Platelet enrichment was performed by adding CaCl2 to the enriched platelets, freezing them at -80°C for 8-10 hours, and then thawing them at 37°C for 5-10 minutes. This freeze-thaw cycle was repeated 10 times. The lysate from the last thawing was centrifuged at 6500 rpm for 15-20 minutes, and the supernatant was retained as platelet lysate (PL). Other steps were the same as in Specific Implementation Method Two.

[0057] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method Three in that the platelet enrichment method is specifically as follows:

[0058] Centrifuge whole blood at 1500-2700 rpm for 15-20 minutes to collect the supernatant plasma, which is platelet-rich plasma. Centrifuge the platelet-rich plasma again at 3500-4300 rpm for 20-25 minutes, and collect the lower layer of platelets. The volume of platelets enriched is 1 / 10 of the whole blood volume. The rest is the same as in Specific Implementation Method 3.

[0059] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods Two to Three in that the volume ratio of PL to exosomes in step two is (40-60):1. Everything else is the same as in Specific Implementation Methods Two to Three.

[0060] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods Two to Three in that the volume ratio of PL to exosomes in step two is 50:1. Everything else is the same as in Specific Implementation Methods Two to Three.

[0061] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods Two to Six in that the concentration of graphene oxide in the Go-sPL suspension in step three is 25-50 μg / mL. Everything else is the same as in Specific Implementation Methods Two to Six.

[0062] Graphene oxide itself possesses antibacterial and anti-inflammatory properties and can promote β-catenin expression, thereby activating the Wnt signaling pathway and aiding in tissue repair. As a nanocarrier encapsulating sPL, it can maintain the activity of growth factors within sPL, increase the loading of growth factors, and achieve a sustained-release effect, more efficiently transporting factors to the damaged site. Simultaneously, sPL can reduce the toxicity of Go and improve biocompatibility; the two work synergistically to enhance the repair effect of gingival recession.

[0063] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods Two to Seven in that the volume ratio of Go-sPL suspension to CREKA peptide in step three is 1:(2-5). Everything else is the same as in Specific Implementation Methods Two to Seven.

[0064] CREKA peptides have the ability to specifically bind fibrin, which allows Go-sPL to target fibrin deposited at the site of gingival inflammation and prolong its retention time at the damaged site, thereby improving the repair effect and efficiency of Go-sPL.

[0065] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0066] Example 1:

[0067] Step 1: Dental Pulp Stem Cell (DPSC) Culture and Exosome Extraction

[0068] (I) Culture of rat DPSCs

[0069] Purified rat dental pulp stem cells (DPSCs) were cultured in complete culture medium at 37°C in a 5% CO2 incubator, and cell growth was observed. Figure 1 and Figure 2 As shown, the cells adhered to the culture medium, exhibiting a fibroblast-like morphology and demonstrating good growth. The complete culture medium was MEM medium containing 10% fetal bovine serum.

[0070] (II) Extraction of exosomes

[0071] When rat DPSCs were cultured to the third generation, and the cells reached 50%-60% adherence, the culture was replaced with the appropriate exosome-free medium for continued culture. When the cells grew to 90%-100%, the culture supernatant was collected into 50mL centrifuge tubes for exosome extraction. The exosome-free medium was prepared as follows: the complete medium was ultracentrifuged at 100,000×g at 4℃ for 16h, and the upper 2 / 3 volume of the medium was collected, which was the exosome-free medium (this step was to remove the exosomes originally present in the complete medium).

[0072] 1. Extraction of exosomes by ultracentrifugation

[0073] (1) Centrifuge the collected supernatant at 4°C and 300×g for 10 min to remove live cells;

[0074] (2) Then, centrifuge at 4°C and 2000×g for 10 min to remove dead cells;

[0075] (3) Then centrifuge at 4℃, 10000×g for 30min to remove cell debris;

[0076] (4) Transfer the supernatant to a 100KD 50ml ultrafiltration tube and centrifuge to collect the concentrate;

[0077] (5) Disinfect the outer wall of the ultrafiltration tube after centrifugation, transfer it to the clean bench, filter the supernatant with a 0.22μm filter, and transfer it to an ultracentrifuge tube;

[0078] (6) Centrifuge at 4℃, 100000×g for 75 min, and retain the precipitate at the bottom of the tube;

[0079] (7) Add PBS to resuspend, centrifuge at 4°C, 100000×g for 75 min, and retain the precipitate at the bottom of the tube;

[0080] (8) Resuspend the precipitate in 150 μL of pre-cooled PBS and store at -80℃ for later use. This is the exosome.

[0081] 2. Western Blot detection of exosome marker proteins

[0082] (1) Mix rat DPSCs and exosome protein samples with 5×buffer at a ratio of 4:1 and incubate in a 90℃ water bath for 8-10 min.

[0083] (2) Prepare the gel according to the instructions of the SDS-PAGE gel preparation kit;

[0084] (3) After the gel solidifies, add the protein marker and exosome protein sample into the spotting well;

[0085] (4) Install the electrophoresis tank, observe the stacking gel for leakage at 70V, adjust to 120V after 30 minutes, and finish electrophoresis in about 60-90 minutes.

[0086] (5) Remove the gel after cutting away the concentrated gel and the useless areas;

[0087] (6) Prepare a PVDF membrane of the same size as the separating gel and activate it with methanol for 1 min;

[0088] (7) Install the transfer device in sequence, from negative electrode to positive electrode: fiber pad, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, fiber pad. Place the transfer device in the transfer tank and operate at 400mA for 30 minutes.

[0089] (8) Remove the PVDF membrane and seal it with 5% skim milk at 25°C for 2 hours. Rinse it twice with TBST for 5 minutes each time.

[0090] (9) Incubate with TSG101 and CD9 antibody at 4°C overnight (about 10 h), and rinse thoroughly with TBST 5 times the next day, 10 min each time;

[0091] (10) Incubate with the corresponding secondary antibody at room temperature for 1 hour, then rinse with TBST 5 times for 10 minutes each time;

[0092] (11) The PVDF membrane was coated with a 1:1 ECL luminescent developing solution, and the experimental results were observed using a gel imaging system.

[0093] See results Figure 3 In the exosome samples, the surface marker proteins TSG101 and CD9 were both positive, while they were not expressed in DPSCs, proving that the exosomes were successfully extracted.

[0094] Step 2: Platelet enrichment methods and preparation of sPL

[0095] 1. Platelet enrichment:

[0096] After anesthetizing the animals, whole blood was collected from rats using blood collection tubes containing sodium citrate anticoagulant.

[0097] The collected whole blood was transferred to the separation chamber at 4°C. After mixing the whole blood, a small amount was taken for counting to calculate the total platelet count. The mixed whole blood was centrifuged at 300×g for 15 min to collect platelet-rich plasma. All the plasma from this centrifugation step was taken, the volume was recorded, and a small amount was taken for platelet counting. The plasma obtained in the previous step was centrifuged again at 1000×g for 20 min. The platelets enriched in the lower layer were collected, ensuring that their volume was 1 / 10 of the whole blood volume. The platelet precipitate adhering to the bottom of the centrifuge tube was fully suspended by pipetting.

[0098] 2. Preparation of SPL:

[0099] (1) Add CaCl2 (20-30mM) to the previously prepared enriched platelets, freeze at -80℃ for 8h, thaw at 37℃ for 5min, repeat freeze-thaw 10 times, and centrifuge the lysate at 3000×g for 15min to obtain platelet lysate (PL).

[0100] (2) DPSCs exosomes are added to the PL obtained in step (1), with the mass ratio of PL to exosomes being 50:1, and further activated to obtain sPL.

[0101] The obtained platelets (PL) and sPL were subjected to ELISA to assess and compare the levels of growth factors promoting gingival fibroblast proliferation, such as VEGF, EGF, PDGF, and TGF-β. The results are shown in Table 1. The results showed that the sPL activated with DPSC exosomes contained higher levels of gingival tissue repair-related growth factors. Therefore, the application of DPSC exosomes can further activate platelets and synergistically produce the desired platelet lysate rich in specific growth factors.

[0102] Table 1. Content of growth factors promoting gingival fibroblast proliferation in sPL under specific treatment.

[0103]

[0104] Step 3: Preparation of the nanomaterial Go-sPL-CREKA

[0105] A Go-sPL-CREKA formulation was constructed by encapsulating sPL with graphene oxide (Go) as a nanocarrier and modifying it with the fibrin-targeting peptide CREKA. The specific method is as follows:

[0106] Graphene oxide was mixed with the sPL prepared in step two, with a final concentration of 30 μg / mL. The mixture was vortexed for 10 minutes to prepare a Go-sPL suspension. Then, CREKA peptide was added, with a volume ratio of 1:2 between the Go-sPL suspension and the CREKA peptide. The mixture was magnetically stirred for 2 hours to obtain the nanomaterial Go-sPL-CREKA.

[0107] The Go-sPL-CREKA nanomaterial requires gentle stirring before use to ensure its homogeneity. All doses of homogenized Go suspension are freshly prepared and heated to 37°C before each injection.

[0108] Example 2: Verification of the interaction effect between graphene oxide (Go) and sPL

[0109] (I) Determination of the optimal working concentration of Go and verification of the reduction of Go toxicity by sPL

[0110] The optimal working concentration of Go was determined using the CCK-8 method, at a concentration of 3 × 10⁻⁶ per well. 3Gingival fibroblasts were seeded into 96-well cell culture plates at a concentration of [number] cells / mL and cultured at 37°C with 5% CO2 for 24 h. After removing the culture medium and washing the cells with PBS, each well was added with culture medium containing working concentrations of Go at 0 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. After incubation for 20 h, the cytotoxic effects of each Go concentration on the cells were measured to determine the optimal working concentration of Go. Results are as follows: Figure 4 As shown, the viability of gingival fibroblasts gradually decreased with increasing Go concentration. When the Go concentration reached 25 μg / mL, the viability of gingival fibroblasts was significantly lower than that of the control group (Go concentration of 0 μg / mL) (p<0.01).

[0111] 3×10 per hole 3 Gingival fibroblasts were seeded into 96-well cell culture plates at a concentration of [number] cells / mL and cultured at 37°C with 5% CO2 for 24 h. After removing the culture medium and washing the cells with PBS, medium containing working concentrations of Go and Go-sPL of 25 μg / mL, 50 μg / mL, and 100 μg / mL were added to each well. After incubation for 20 h, the cytotoxic effects of each concentration of Go and Go-sPL on the cells were detected. The results are as follows: Figure 5 As shown, compared with the control group, the cell viability of the 25 μg / mL and 50 μg / mL Go-sPL groups was comparable, while the cell viability of all Go groups and the 100 μg / mL Go-sPL group was significantly reduced (p<0.01), indicating that sPL can alleviate the toxic effect of Go on gingival fibroblasts.

[0112] (II) Verification of the sustained-release effect of Go on cytokines

[0113] Two mL of Go-sPL at concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL were placed in test tubes containing 10 mL of PBS and incubated on a shaker at 37 °C and 100 rpm. Two mL of PBS solution was collected at different time points (1 d, 3 d, 5 d, 7 d, and 14 d) and stored at –20 °C. At each time point, the concentrations of VEGF and PDGF in the collected PBS were quantified using the corresponding enzyme-linked immunosorbent assay (ELISA) kit to verify the sustained-release effect. Results are as follows: Figure 6 and 7 As shown in the figure Indicates sPL, This indicates a Go-sPL concentration of 5 μg / mL. This indicates 10 μg / mL Go-sPL. This indicates 25 μg / mL Go-sPL. This indicates 50 μg / mL Go-sPL. This indicates a Go-sPL concentration of 100 μg / mL. During the first 7 days, sPL released most of the VEGF and PDGF, while Go-sPL controlled the release of growth factors within it. The rate of growth factor release decreased with increasing Go concentration. In the following 7 days, the release of growth factors from sPL significantly decreased, while 25, 50, and 100 μg / mL Go-sPL maintained a substantial release of growth factors.

[0114] In summary, Go-sPL exhibits optimal sustained-release efficacy at concentrations of 25-100 μg / mL. However, Go exhibits gingival fibroblast toxicity at concentrations exceeding 25 μg / mL, with the toxicity increasing at higher concentrations. Furthermore, the results show that sPL can reduce the toxicity of Go to gingival fibroblasts, achieving significant attenuation at concentrations of 25 and 50 μg / mL. Therefore, limiting the working concentration of Go-sPL to 25-50 μg / mL ensures optimal sustained-release efficacy without toxicity to gingival fibroblasts.

[0115] Example 3: Verification of the effect of the nanomaterial Go-sPL-CREKA prepared in Example 1 on the repair of gingival recession

[0116] (I) Cellular Experiments

[0117] Gingival fibroblasts were cultured in MEM medium containing 5% (v / v) PL, MEM medium containing 5% (v / v) sPL, MEM medium containing 5% (v / v) Go-sPL, and MEM medium containing 5% (v / v) Go-sPL-CREKA in a 37°C, 5% CO2 incubator. After the cells were passaged to the 5th generation, cell proliferation was detected by CCK-8 assay and the expression of cell proliferation-related proteins was detected by Western blot. The different culture media were compared.

[0118] Gingival fibroblasts play a crucial role in the repair of gingival recession; therefore, in vitro cell experiments were conducted to evaluate the efficacy of the nanomaterial Go-sPL-CREKA. The CCK-8 assay was used to detect the proliferation of gingival fibroblasts, and the results are as follows: Figure 8 As shown, Figure 8 ● represents PL, ▼ represents sPL, ■ represents Go-sPL, and ▲ represents Go-sPL-CREKA. Figure 8 The results showed that, compared with the control group (PL), the OD values ​​of the sPL, Go-sPL and Go-sPL-CREKA groups were all increased, indicating that all three preparations could promote the proliferation of gingival fibroblasts, with the Go-sPL-CREKA group showing the best effect.

[0119] Western blot analysis was performed to detect the expression of proteins related to gingival fibroblast proliferation and gingival repair. The expression of DKK1 protein was as follows: Figure 9 As shown, the expression of β-atenin protein is as follows: Figure 10 As shown, "*" indicates a significant difference compared to the control group (P<0.05), "**", "***" and "****" indicate extremely significant differences compared to the control group (P<0.01), and no label indicates no statistical difference compared to the control group (P>0.05). The results showed that, compared to PL, sPL could inhibit DKK1 protein expression, thereby promoting β-atenin protein expression and activating the Wnt / β-atenin signaling pathway. Go could upregulate β-atenin protein expression, and its synergistic effect with sPL further promoted gingival fibroblast proliferation, contributing to tissue repair and regeneration.

[0120] The occurrence of gingival recession is closely related to the growth of periodontal pathogens. Therefore, periodontal tissues from rats with gingival recession were used to test their antibacterial properties. The changes in bacterial viability after Go treatment are shown in the following figures. Figure 11 As shown in the results, compared with the simple sPL group, both the Go-sPL and Go-sPL-CREKA groups had better antibacterial effects, proving that Go has a certain anti-periodontal pathogenic effect and can help repair gingival recession.

[0121] (II) Zoological Experiments

[0122] A gingival recession model was established in rats by ligating both maxillary first molars with 0.2 mm orthodontic ligation wire for 2 weeks. The rats were then divided into four groups for treatment: Group 1 was the control group (saline sham treatment); Group 2 received sPL injection; Group 3 received Go-sPL injection; and Group 4 received Go-sPL-CREKA injection. All four groups received injections at the same dosage, time, and frequency, injected into the gingival recession site every 7 days for a total of 3 treatments, constituting one course of treatment. Seven days after the last treatment, the rats were sacrificed, and whole blood was collected. Fresh blood was used for hematological and biochemical analysis to observe changes in various indicators in the blood of the four groups and to test the toxicity of graphene oxide to the animals. Changes in gingival tissue were observed, and the gingival tissue was cryopreserved for nucleic acid protein level testing and comparison of treatment effects.

[0123] Various formulations for animal biosafety testing, such as Figure 12 show, Figure 12● represents the control group, ■ represents the sPL injection group, ▲ represents the Go-sPL injection group, and ▼ represents the Go-sPL-CREKA injection group. The body weight of rats in all groups increased during the experimental phase, but there was no statistically significant difference in body weight among the three treatment groups compared to the control group (P>0.05). The effects of various injection preparations on rat hematological and biochemical parameters are shown in Table 2. There were no statistically significant differences in whole blood and serum analysis parameters among the three treatment groups compared to the control group. These two results indicate that Go and CREKA have no toxic effects on rats. The expression of COL-1, COL-3, and VEGF genes related to soft tissue repair and regeneration was detected using qRT-PCR. Figures 13-15 As shown, "*" indicates a significant difference compared to the control group (P<0.05), "**" and "***" indicate an extremely significant difference compared to the control group (P<0.01) (P<0.001), and no marker indicates no statistical difference compared to the control group (P>0.05).

[0124] Compared with the control group, the expression of COL-1 and VEGF was upregulated in all three treatment groups, and the differences were significant. The expression of COL-3 was significantly upregulated in the Go-sPL and Go-sPL-CREKA treatment groups. The results indicate that all three treatment groups have a certain therapeutic effect on gingival recession repair, which may be achieved by increasing the expression of COL-1, COL-3 and VEGF to promote collagen and angiogenesis, thereby promoting gingival growth. Among them, Go-sPL-CREKA treatment showed the best effect.

[0125] Table 2 Effects of the injectable formulation on hematological and biochemical parameters in rats

[0126]

[0127]

[0128] In Table 2, WBC represents white blood cell count; RBC represents red blood cell count; Hgb represents hemoglobin; Hct represents hematocrit; MCV represents mean corpuscular volume; MCH represents mean corpuscular hemoglobin; MCHC represents mean corpuscular hemoglobin concentration; PLT represents platelet count; ALT represents alanine aminotransferase; AST represents aspartate aminotransferase; ALP represents alkaline phosphatase; GGT represents gamma-glutamyl transferase; DB represents direct bilirubin; TB represents total bilirubin; BUN represents blood urea nitrogen; Crea represents creatinine; CPK represents creatine phosphokinase; CK-MB represents creatine kinase-MB; TP represents total protein; TG represents triglycerides; CHO represents cholesterol; HDL represents high-density lipoprotein; and LDL represents low-density lipoprotein.

Claims

1. An sPL nanomaterial for repairing gingival recession, characterized in that... The nanomaterial is made from platelet lysis fluid PL, exosomes, graphene oxide Go, and fibrin-targeting peptide CREKA, with the exosomes extracted from dental pulp stem cells; The sPL is prepared by mixing platelet lysis fluid PL with exosomes; the graphene oxide Go serves as a nanocarrier to encapsulate the sPL and is modified with fibrin-targeting peptide CREKA to construct Go-sPL-CREKA. The specific method for preparing the platelet lysis buffer PL is as follows: Platelet enrichment was performed by adding CaCl2 to the enriched platelets, freezing them at -80℃ for 8-10 h, and then thawing them at 37℃ for 5-10 min. This freeze-thaw cycle was repeated 10 times. The lysate from the last thaw was centrifuged at 6500 rpm for 15-20 min, and the supernatant was retained, which is the platelet lysate PL.

2. The method for preparing sPL nanomaterials for repairing gingival recession as described in claim 1, characterized in that... The method includes the following steps: Step 1: The purified rat dental pulp stem cells were cultured in complete culture medium to the third generation. When the cells adhered and grew to 50%-60%, the culture was replaced with the appropriate exosome-free culture medium and cultured for another 90%-100% of the cells. The culture supernatant was collected and the exosomes were extracted. Step 2: Prepare platelet lysis buffer (PL), mix PL with exosomes to obtain sPL; Step 3: Mix graphene oxide with the sPL prepared in Step 2 to obtain Go-sPL suspension, then add fibrin targeting peptide CREKA, stir for 2-3 hours to prepare nanomaterial Go-sPL-CREKA; The specific method for preparing platelet lysis buffer (PL) in step two is as follows: Platelet enrichment was performed by adding CaCl2 to the enriched platelets, freezing them at -80℃ for 8-10 h, and then thawing them at 37℃ for 5-10 min. This freeze-thaw cycle was repeated 10 times. The lysate from the last thaw was centrifuged at 6500 rpm for 15-20 min, and the supernatant was retained, which is the platelet lysate PL.

3. The method for preparing sPL nanomaterials for repairing gingival recession according to claim 2, characterized in that, The specific method for platelet enrichment is as follows: Centrifuge whole blood at 1500-2700 rpm for 15-20 min to collect the upper plasma layer, which is platelet-rich plasma. Centrifuge the platelet-rich plasma again at 3500-4300 rpm for 20-25 min and collect the lower layer of platelets. The volume of the enriched platelets is 1 / 10 of the whole blood volume.

4. The method for preparing sPL nanomaterials for repairing gingival recession according to claim 2, characterized in that, In step two, the volume ratio of PL to exosomes is (40-60):

1.

5. The method for preparing sPL nanomaterials for repairing gingival recession according to claim 2 or 3, characterized in that, Step 3: The concentration of graphene oxide in the Go-sPL suspension is 25-50 μg / mL.

6. The method for preparing sPL nanomaterials for repairing gingival recession according to claim 5, characterized in that, In step three, the volume ratio of Go-sPL suspension to CREKA peptide is 1:(2-5).

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