CN116637201B - SPL nano material for repairing gingival recession and preparation method thereof - Google Patents
SPL nano material for repairing gingival recession and preparation method thereofInfo
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Abstract
一种用于修复牙龈退缩的sPL纳米材料及其制备方法，涉及牙龈退缩修复材料领域，尤其涉及一种用于修复牙龈退缩的sPL纳米材料及其制备方法。该纳米材料是由血小板裂解液、外泌体、氧化石墨烯和CREKA多肽制成，所述外泌体提取自牙髓干细胞。方法：步骤一：外泌体的提取；步骤二：sPL的制备；步骤三：纳米材料Go‑sPL‑CREKA的制备。本发明的纳米材料能够促进牙龈成纤维细胞增殖，提高牙龈退缩修复相关生长因子含量，加强牙龈退缩的修复效果。本发明用于修复牙龈退缩。
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
The invention relates to the field of gingival retraction and repair materials, in particular to an sPL nano material for repairing gingival retraction and a preparation method thereof.
Background
Periodontal disease is one of the most common diseases worldwide. In addition to periodontitis and gingivitis, there are other diseases and changes that can affect periodontal tissue. However, periodontitis has the greatest impact on adult oral health and public health. Periodontitis is a chronic, inflammatory, non-infectious disease that affects all parts of the periodontal tissue and causes irreversible damage to the periodontal tissue. The prevalence of this disease increases gradually with age. Periodontitis becomes a more prominent problem under the increasing prominence of aging in China society.
Gingival Recession (GR) is a common symptom of periodontitis, and refers to that the gingival margin of teeth is located in the direction of the root tip of the cementum field, or simultaneously with the recession of the papilla between teeth, which causes exposure of the tooth root and affects the beauty, and serious gingival recession may have a certain amount of alveolar bone absorption and loss of attachment. In recent years, problems associated with restoration and aesthetics due to gingival recession have been emphasized. The gingival retraction and periodontitis are mutually influenced, the gingival retraction possibly causes periodontitis, the periodontitis can directly lead to the gingival retraction, if the periodontitis is also caused while the gingival retraction, the situation of the gingival retraction can be aggravated, the absorption of alveolar bone is accelerated, the mutual relation is vicious, the gingival attachment level is reduced, and further, the undesirable symptoms such as the increment of tooth gaps, the exposure of tooth roots, the loosening of teeth and the like appear. Therefore, attention needs to be paid, and as the root of the tooth is exposed due to serious gingival recession, satisfactory effect is difficult to achieve by means of self-repair, and the repair treatment is clinically carried out by usually carrying out operation, but the method has certain limitation and is more traumatic.
In recent years, tissue engineering technology based on stem cells has become an important and hot point of research in the oral field, and a currently common method for repairing gingival recession is to add growth factors into materials. However, the addition of one growth factor has single effect, short half-life period and poor prevention and repair effects, and if a plurality of growth factors are added, the technical requirement is higher and the operation difficulty is high.
Disclosure of Invention
The invention aims to solve the problems of single effect of growth factors, poor prevention and repair effects of the existing method, and provides an sPL nano material for repairing gingival recession and a preparation method thereof.
The sPL nanometer material for repairing gingival recession is prepared from platelet lysate, exosomes, graphene oxide and CREKA polypeptide, wherein the exosomes are extracted from dental pulp stem cells.
The invention relates to a preparation method of sPL nanometer material for repairing gingival recession, which comprises the following steps:
Step one, extracting exosomes
Culturing the purified rat dental pulp stem cells to the third generation by using a complete culture medium, changing the culture medium into a corresponding culture medium without exosomes to continue culturing when the cell wall-attached growth reaches 50% -60%, collecting culture supernatant when the cell wall-attached growth reaches 90% -100%, and extracting exosomes;
Step two, preparation of sPL
Preparing platelet lysate PL, mixing PL with exosomes to obtain sPL;
step three, preparation of nano material Go-sPL-CREKA
Mixing graphene oxide with sPL prepared in the second step to obtain a Go-sPL suspension, adding CREKA peptide, and stirring for 2-3 hours to prepare the nano-material Go-sPL-CREKA.
Further, the preparation method of the platelet lysate PL in the second step specifically comprises the following steps:
Platelet enrichment is carried out, caCl 2 is added into the blood platelets after the platelet enrichment, the blood platelets are frozen at-80 ℃ for 8-10 hours, then thawed at 37 ℃ for 5-10 minutes, repeated freeze thawing is carried out for 10 times, finally dissolved lysate is centrifuged at 6500rpm for 15-20 minutes, and supernatant is reserved, thus obtaining platelet lysate PL.
The platelet enrichment method specifically comprises the following steps:
centrifuging whole blood at 1500-2700rpm for 15-20min to collect upper plasma, namely platelet-rich plasma, centrifuging the platelet-rich plasma again at 3500-4300rpm for 20-25min, and collecting lower layer of enriched platelets, wherein the volume of the enriched platelets is 1/10 of that of whole blood.
Further, the volume ratio of PL to exosome in the second step is (40-60): 1.
Further, the concentration of graphene oxide in the suspension of step three Go-sPL is 25-50 μg/mL.
Further, the volume ratio of the Go-sPL suspension to CREKA peptide in step three is 1 (2-5).
According to the invention, graphene oxide (Go) is used as a nano-carrier to wrap specific activated sPL, and polypeptide CREKA is modified to prepare a novel nano-material for promoting cell regeneration and tissue repair, and the novel nano-material is used for improving and treating gingival recession. By taking functions of promoting proliferation and differentiation of gingival fibroblasts and the like as an entry point, a sPL preparation method capable of increasing secretion of growth factors of VEGF, EGF, PDGF, TGF-beta and the like which promote proliferation and tissue repair of the gingival fibroblasts is sought. The sPL and Go are combined and modified by CREKA to directionally activate gingival fibroblasts, promote proliferation and self-renewal capacity of the gingival fibroblasts, target fibrin deposited on inflammatory parts, strengthen antibacterial capacity and restoration effect of gingival tissues, and provide technical support and treatment thought for gingival retraction clinical research of new sPL nano materials.
The invention has the beneficial effects that:
1. The invention adopts a two-step centrifugation method, wherein the first step is centrifugation at 1500-2700rpm for 15min, the blood plasma and blood cells are separated by using low rotation speed, the blood platelets are ensured to stay in the blood plasma and not to sink into the blood cells, the whole blood plasma centrifuged in the first step is collected for a second centrifugation, the centrifugation condition is 3500-4300rpm for 20min, so that the blood platelets are settled in the bottom of a tube and the bottom blood plasma as much as possible, the upper blood platelet content is lower, and the blood platelet concentration efficiency is enhanced.
2. The sPL is prepared by adding DPSCs exosomes into PL for further activation, can promote fibroblast proliferation, improve the content of growth factors related to gingival tissue repair, and can inhibit DKK1 expression in gingival fibroblasts by synergistic effect with exosomes, activate Wnt signal paths, increase the concentration of effective factors and enhance gingival retraction and repair effects.
3. The graphene oxide has antibacterial and anti-inflammatory effects, can promote the expression of beta-catenin, further activates a Wnt signal path, and is beneficial to tissue injury repair. As nano-carrier to wrap sPL, it can promote the growth factor in sPL to keep activity, raise the loading quantity of growth factor and reach slow release function, more effectively transport factor to damaged position, at the same time sPL can reduce the toxic action of Go, raise biological safety, and both can cooperatively strengthen the restoration effect of gingival retraction.
4. The CREKA peptide has the capability of specifically binding with fibrin, can lead the Go-sPL to target the fibrin deposited on the gingival inflammation part, prolong the retention time of the fibrin on the injury part and improve the repair effect and efficiency of the Go-sPL.
According to the Go-sPL-CREKA nano material, the characteristics of relatively rich growth factors and nutrient components related to gingival tissue repair in sPL are utilized, the Go nano material is wrapped in a Go nano carrier, and by adding DPSCs exosomes, gingival fibroblast proliferation and differentiation factors (such as VEGF, EGF, PDGF, TGF-beta and the like) are directionally activated, and the targeting repair effect of CREKA peptide is combined, so that the proliferation and the characteristic maintenance of gingival fibroblast are facilitated, the antibacterial capability is enhanced, and the biological safety is good.
The invention can improve the treatment effect in the gingival retraction and restoration process, and provides a new thought and a new method for clinical researches on periodontal tissue diseases such as gingival retraction.
Drawings
FIG. 1 shows growth of rats at DPSCs passage 1;
FIG. 2 shows growth of rats at DPSCs th passage;
FIG. 3 is an identification of exosome markers;
FIG. 4 is a graph showing the detection of Go's toxic effects on gingival fibroblasts;
FIG. 5 is the effect of sPL on Go cytotoxicity;
FIG. 6 shows the detection of the slow release effect of Go on the cytokine VEGF in sPL;
FIG. 7 shows the detection of the slow release effect of Go on the cytokine PDGF in sPL;
FIG. 8 shows proliferation of gingival fibroblast (OD at 450 nm);
FIG. 9 shows the DKK1 protein expression of gingival fibroblast proliferation;
FIG. 10 shows the expression of β -atenin protein from gingival fibroblast proliferation;
FIG. 11 shows the change in bacterial activity after graphene oxide treatment;
FIG. 12 shows the change in body weight of rats in each group;
FIG. 13 shows gingival tissue repair COL-1 gene expression;
FIG. 14 shows gingival tissue repair COL-2 gene expression;
FIG. 15 shows gingival tissue repair VEGF gene expression.
Detailed Description
The technical scheme of the invention is not limited to the specific embodiments listed below, and also includes any combination of the specific embodiments.
In a first embodiment, the sPL nanomaterial for restoring gingival recession in the present embodiment is prepared from platelet lysate, exosomes, graphene oxide and CREKA polypeptide, wherein the exosomes are extracted from dental pulp stem cells.
In the embodiment, graphene oxide (Go) is used as a nano-carrier to wrap specific activated sPL, and polypeptide CREKA is modified to prepare a novel nano-material for promoting cell regeneration and tissue repair, and the novel nano-material is used for improving and treating gingival recession. By taking functions of promoting proliferation and differentiation of gingival fibroblasts and the like as an entry point, a sPL preparation method capable of increasing secretion of growth factors of VEGF, EGF, PDGF, TGF-beta and the like which promote proliferation and tissue repair of the gingival fibroblasts is sought. The sPL and Go are combined and modified by CREKA to directionally activate gingival fibroblast, promote proliferation and self-renewal capacity of gingival fibroblast, target fibrin deposited on inflammation part and strengthen antibacterial capacity and repairing effect of gingival tissue
The preparation method of the sPL nanometer material for repairing gingival recession comprises the following steps of:
Step one, extracting exosomes
Culturing the purified rat dental pulp stem cells to the third generation by using a complete culture medium, changing the culture medium into a corresponding culture medium without exosomes to continue culturing when the cell wall-attached growth reaches 50% -60%, collecting culture supernatant when the cell wall-attached growth reaches 90% -100%, and extracting exosomes;
Step two, preparation of sPL
Preparing platelet lysate PL, mixing PL with exosomes to obtain sPL;
step three, preparation of nano material Go-sPL-CREKA
Mixing graphene oxide with sPL prepared in the second step to obtain a Go-sPL suspension, adding CREKA peptide, and stirring for 2-3 hours to prepare the nano-material Go-sPL-CREKA.
The sPL in the second embodiment is prepared by adding DPSCs exosomes into PL for further activation, can promote proliferation of fibroblasts, improve the content of growth factors related to gingival tissue repair, and can inhibit DKK1 expression in gingival fibroblasts by synergistic effect with exosomes, activate Wnt signal paths, increase the concentration of effective factors and enhance gingival retraction and repair effects.
The third embodiment is different from the second embodiment in that the preparation method of the platelet lysate PL in the second step specifically comprises the following steps:
Platelet enrichment is carried out, caCl 2 is added into the blood platelets after the platelet enrichment, the blood platelets are frozen at-80 ℃ for 8-10 hours, then thawed at 37 ℃ for 5-10 minutes, repeated freeze thawing is carried out for 10 times, finally dissolved lysate is centrifuged at 6500rpm for 15-20 minutes, and supernatant is reserved, thus obtaining platelet lysate PL. The other is the same as in the second embodiment.
The fourth embodiment is different from the third embodiment in that the platelet enrichment method specifically includes:
Centrifuging whole blood at 1500-2700rpm for 15-20min to collect upper plasma, namely platelet-rich plasma, centrifuging the platelet-rich plasma again at 3500-4300rpm for 20-25min, and collecting lower layer of enriched platelets, wherein the volume of the enriched platelets is 1/10 of that of whole blood. The other is the same as in the third embodiment.
In the fifth embodiment, the difference between the second embodiment and the third embodiment is that the volume ratio of PL to exosome in the second step is (40-60): 1. The other is the same as in one of the second to third embodiments.
In the sixth embodiment, the difference between the second embodiment and the third embodiment is that the volume ratio of PL to exosome in the second step is 50:1. The other is the same as in one of the second to third embodiments.
In a seventh embodiment, the concentration of graphene oxide in the suspension of step three Go-sPL is 25-50 μg/mL, which is different from one of the second to sixth embodiments. The others are the same as in the second to sixth embodiments.
The graphene oxide has antibacterial and anti-inflammatory effects, can promote the expression of beta-catenin, further activates a Wnt signal path, and is beneficial to tissue injury repair. As nano-carrier to wrap sPL, it can promote the growth factor in sPL to keep activity, raise the loading quantity of growth factor and reach slow release function, more effectively transport factor to damaged position, at the same time sPL can reduce the toxic action of Go, raise biological safety, and both can cooperatively strengthen the restoration effect of gingival retraction.
In the eighth embodiment, the difference between the second embodiment and the seventh embodiment is that the volume ratio of the Go-sPL suspension to the CREKA peptide in the third step is 1 (2-5). The others are the same as in one of the second to seventh embodiments.
The CREKA peptide has the capability of specifically binding with fibrin, can lead the Go-sPL to target the fibrin deposited on the gingival inflammation part, prolong the retention time of the fibrin on the injury part and improve the repair effect and efficiency of the Go-sPL.
The following examples of the present invention are described in detail, and are provided by taking the technical scheme of the present invention as a premise, and the detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.
Example 1:
step one, dental Pulp Stem Cell (DPSCs) culture and exosome extraction
Culture of rats DPSCs
The purified rat dental pulp stem cells DPSCs were cultured in a 37 ℃ and 5% CO 2 incubator with complete medium, and the cell growth conditions were observed as shown in fig. 1 and 2, and it was seen that the cells grew on the wall and were in a fibroblast-like morphology with good growth conditions. The complete medium is MEM medium containing 10% fetal bovine serum.
Extraction of exosomes
When the rat DPSCs is cultivated to the third generation, the culture medium is changed into a corresponding exosome-free culture medium to continue the cultivation when the cell is attached to 50% -60%, and when the cell is grown to 90% -100%, the culture supernatant is collected into a 50mL centrifuge tube to be used for extracting exosome. The preparation method of the exosome-free culture medium comprises the steps of ultracentrifugation of the complete culture medium at 4 ℃ and 100000 Xg for 16 hours, and collection of 2/3 volume of the culture medium at the upper layer, namely the exosome-free culture medium (the function of the step is to remove the original exosome in the complete culture medium).
1. Extraction of exosomes by ultracentrifugation
(1) The collected supernatant was centrifuged at 300 Xg for 10min at 4℃to remove living cells;
(2) Dead cells were then removed by centrifugation at 2000 Xg for 10min at 4 ℃;
(3) Then, cell debris was removed by centrifugation at 10000 Xg for 30min at 4 ℃;
(4) Transferring the supernatant to a 50ml ultrafiltration tube with 100KD, and centrifuging to collect a concentrated solution;
(5) Sterilizing the outer wall of the centrifuged supernatant ultrafiltration tube, transferring to an ultra-clean bench, filtering the supernatant by a 0.22 mu m filter, and transferring to an ultra-centrifuge tube;
(6) Ultracentrifugation at 4 ℃ and 100000 Xg for 75min, and retaining the tube bottom sediment;
(7) Adding PBS for resuspension, centrifuging at 4 ℃ and 100000 Xg for 75min, and keeping the sediment at the bottom of the tube;
(8) Re-suspending the sediment with 150 mu L of precooled PBS, and reserving at-80 ℃ to obtain the exosomes.
2. Western Blot detection of exosome marker proteins
(1) Uniformly mixing a rat DPSCs and an exosome protein sample with a 5 Xbuffer according to a ratio of 4:1, and then carrying out water bath at 90 ℃ for 8-10min;
(2) Preparing a gel according to the instruction book of the SDS-PAGE gel preparation kit;
(3) After the gel is fixed, adding a protein Marker and an exosome protein sample into the sample application hole;
(4) Installing an electrophoresis tank, observing whether the concentrated glue leaks samples or not at 70V, adjusting 120V after 30min, and finishing electrophoresis for about 60-90 min;
(5) Removing the concentrated gel and useless areas, and then taking down the gel;
(6) Preparing a PVDF film with the same size as the separation gel, and activating methanol for 1min;
(7) Sequentially installing a transfer printing device, namely a fiber pad, three layers of filter paper, gel, PVDF (polyvinylidene fluoride) films, three layers of filter paper and the fiber pad from a negative electrode to a positive electrode, and putting the transfer printing device into a film transfer groove to work for 30min at 400 mA;
(8) Taking out PVDF membrane, sealing with 5% skimmed milk at 25deg.C for 2 hr, rinsing with TBST for 2 times each for 5min;
(9) Incubation with TSG101 and CD9 antibody at 4 ℃ overnight (about 10 h), rinsing with TBST 5 times per day for 10min;
(10) Incubating with the corresponding secondary antibody for 1h at room temperature, and rinsing with TBST for 5 times for 10min each time;
(11) PVDF film was fully coated with 1:1 ECL luminescence developer, and the experimental results were observed by a gel imaging system.
The results are shown in FIG. 3, in the exosome samples, both surface marker proteins TSG101 and CD9 were positive, but were not substantially expressed in DPSCs, demonstrating successful exosome extraction.
Step two, platelet enrichment method and sPL preparation
1. Platelet enrichment:
after the animals are anesthetized, the whole blood of the rats is collected by utilizing a blood collection tube containing sodium citrate anticoagulant;
Transferring the collected whole blood to a separation chamber at 4 ℃, uniformly mixing the whole blood, taking a small amount of the whole blood to count, calculating the total amount of platelets in the whole blood, centrifuging the uniformly mixed whole blood at 300 Xg for 15min to collect platelet-rich plasma, taking out the whole blood after centrifugation, recording the volume, extracting a small amount of the platelet-rich plasma, centrifuging the obtained blood after the step for 20min again at 1000 Xg, reserving the platelet-rich plasma at the lower layer, ensuring that the volume of the platelet-rich plasma is 1/10 of the volume of the whole blood, and blowing the platelet sediment with the bottom fully suspended at the bottom of a centrifuge tube by using a pipette.
2. Preparation of sPL:
(1) CaCl 2 (20-30 mM) was added to the previously prepared and enriched platelets, and after storage at-80℃for 8 hours, thawing was performed at 37℃for 5min, repeated freeze thawing was performed 10 times, and the lysate of the last solubilization was centrifuged at 3000 Xg for 15min to obtain Platelet Lysate (PL).
(2) Adding DPSCs exosomes into the PL obtained in the step (1), wherein the mass ratio of the PL to the exosomes is 50:1, and further activating to obtain sPL.
ELISA was performed on PL and sPL obtained above to evaluate and compare the content of growth factors such as VEGF, EGF, PDGF and TGF-. Beta.that promote gingival fibroblast proliferation, and the results are shown in Table 1. The results show that in sPL activated by the DPSCs exosomes, the released gingival tissue repair related growth factors are higher, so that the application of DPSCs exosomes can further activate and cooperate with platelets to obtain the required platelet lysate rich in specific growth factors.
TABLE 1 growth factor content for gingival fibroblast proliferation in sPL under specific treatment
Step three, preparation of nano material Go-sPL-CREKA
And (3) wrapping sPL with graphene oxide (Go) serving as a nano-carrier, and modifying with fibrin targeting peptide CREKA to construct a Go-sPL-CREKA preparation. The specific method comprises the following steps:
Mixing graphene oxide with sPL prepared in the second step, wherein the final concentration of the graphene oxide is 30 mug/mL, carrying out vortex oscillation for 10 minutes to prepare a Go-sPL suspension, adding CREKA peptide, wherein the volume ratio of the Go-sPL suspension to the CREKA peptide is 1:2, and magnetically stirring for 2 hours to obtain the nano material Go-sPL-CREKA.
The nanomaterial Go-sPL-CREKA needs to be gently stirred before use to ensure homogeneity of the nanomaterial. All doses of homogeneous Go suspension were freshly prepared, requiring heating at 37 ℃ before each injection use.
Example 2 interaction Effect verification of graphene oxide (Go) with sPL
Determination of optimal working concentration of Go and verification of sPL reduced Go toxicity
Determining the optimal working concentration of Go by using a CCK-8 method, inoculating gingival fibroblasts into a 96-well cell culture plate according to the concentration of 3X10 3 per mL of each well, placing the culture plate into a37 ℃ 5% CO 2 constant-temperature incubator for culturing for 24 hours, removing the culture medium, respectively adding culture medium containing Go with the working concentration of 0 mug/mL, 5 mug/mL, 10 mug/mL, 25 mug/mL, 50 mug/mL and 100 mug/mL into each well after PBS is used for cleaning the cells, and detecting the toxic effect of each concentration Go on the cells after incubation for 20 hours so as to determine the optimal working concentration of the Go. As a result, as shown in FIG. 4, the viability of gingival fibroblasts was gradually decreased with an increase in the Go concentration, and when the Go concentration reached 25. Mu.g/mL, the viability of gingival fibroblasts was extremely significantly decreased (p < 0.01) compared to the control group (Go concentration 0. Mu.g/mL group).
Gingival fibroblasts were inoculated into 96-well cell culture plates at a concentration of 3×10 3 per mL, placed in a 37 ℃ 5% CO 2 incubator for culture for 24 hours, medium was removed, PBS was added to each well after washing the cells, medium containing Go and Go-sPL at working concentrations of 25 μg/mL, 50 μg/mL, 100 μg/mL was added, and after incubation for 20 hours, the toxic effects of each concentration Go and Go-sPL on the cells were examined, and as a result, as shown in fig. 5, the cell viability was comparable for the Go-sPL groups at 25 μg/mL and 50 μg/mL compared to the control group, whereas the cell viability was significantly reduced for the Go groups at all concentrations and 100 μg/mLGo-sPL groups (p < 0.01), indicating that sPL can alleviate the toxic effects of Go on gingival fibroblasts.
(II) verification of the sustained Release action of Go on cytokines
Go-sPL at a concentration of 2 mLGo. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, respectively, was placed in a tube containing 10mL PBS and incubated on a shaker at 37℃at 100 r/min. 2mL of PBS solution was collected at various time points (1 d, 3d, 5d, 7d, and 14 d) and stored at-20 ℃. At each time point, the concentrations of VEGF and PDGF in the collected PBS were quantified using the corresponding enzyme-linked immunosorbent assay kit to verify the slow release effect. The results are shown in FIGS. 6 and 7, in whichThe expression sPL is used to indicate that,Representing 5. Mu.g/mL Go-sPL,Representing 10. Mu.g/mL Go-sPL,Represents 25. Mu.g/mL Go-sPL,Representing 50. Mu.g/mL Go-sPL,Representing 100. Mu.g/mL Go-sPL. In the first 7 days, sPL released most of the VEGF and PDGF, while Go-sPL exerted a controlled release effect on its growth factors. The growth factor release rate decreases with increasing Go concentration. During the next 7 days, the release of growth factors from sPL was significantly reduced, while 25, 50, 100 μg/mL Go-sPL maintained the release of a large amount of growth factors.
In summary, go-sPL has the best factor slow-release effect when the concentration is 25-100 mug/mL, but Go has gingival fibroblast toxicity after the concentration reaches 25 mug/mL, and the greater the concentration is, the stronger the toxicity is, meanwhile, the detection result shows that sPL can reduce the toxic effect of Go on gingival fibroblast, and can achieve good attenuation effect when the concentration is 25 and 50 mug/mL. Therefore, the working concentration of Go-sPL is limited to 25-50 μg/mL, which is optimal for the slow release of the effective factors and is not detrimental to gingival fibroblasts.
EXAMPLE 3 verification of the gingival recession repair Effect of the nanomaterial Go-sPL-CREKA prepared in example 1
Cytological experiments
The gingival fibroblast cells were cultured in a MEM medium containing 5% (v/v) PL, a MEM medium containing 5% (v/v) sPL, a MEM medium containing 5% (v/v) Go-sPL and a MEM medium containing 5% (v/v) Go-sPL-CREKA in a 37℃and 5% CO 2 incubator, respectively, and when the cells were transferred to 5 passages, the cell proliferation was detected by CCK-8 and the cell proliferation-related protein expression was detected by Western blot, and the different media were compared.
Gingival fibroblasts play a critical role in restoration of gingival recession, so in vitro cell experiments are performed to evaluate the effect of the nanomaterial Go-sPL-CREKA. CCK-8 experiments were performed to examine proliferation of gingival fibroblasts, and the results are shown in FIG. 8, wherein +.i. in FIG. 8 represents PL, and wherein "x" represents sPL, ■ represents Go-sPL, and "A" represents Go-sPL-CREKA. FIG. 8 shows that the OD values of the sPL, go-sPL and Go-sPL-CREKA groups were all increased compared to the control group (PL), indicating that the three formulations all promoted proliferation of gingival fibroblasts, with the Go-sPL-CREKA group being the most effective.
Western blot examined gingival fibroblast proliferation and gingival repair related protein expression, DKK1 protein expression as shown in fig. 9, and β -atenin protein expression as shown in fig. 10, "indicates significant differences (P < 0.05) from the control group," "and" "indicate significant differences (P < 0.01) from the control group (P < 0.001) (P < 0.0001), no sign indicates no statistical differences (P > 0.05) from the control group. The result shows that compared with PL, sPL can inhibit DKK1 protein expression, promote beta-atenin protein expression, activate Wnt/beta-atenin signal path, and Go can up regulate beta-atenin protein expression, and further promote gingival fibroblast proliferation by synergistic action with sPL, thereby being beneficial to tissue repair and regeneration.
The occurrence of gingival retraction is closely related to the growth of periodontal pathogenic bacteria, so that the periodontal tissue of a rat after gingival retraction and modeling is taken for antibacterial performance detection, the change condition of bacterial activity after Go treatment is shown as shown in figure 11, and the result shows that compared with a simple sPL group, the Go-sPL and Go-sPL-CREKA both have better antibacterial effects, and the Go has certain periodontal pathogenic bacteria resisting effect and certain help to the restoration of gingival retraction.
(II) animal experiments
The rat bilateral maxillary first molar was ligated with 0.2mm orthodontic ligature wire for 2 weeks, and a gingival retraction model was established. The model rats were treated in four groups, the first group was a control group (saline sham treatment group), the second group was a sPL preparation injection group, the third group was a Go-sPL preparation injection group, and the fourth group was a Go-sPL-CREKA preparation injection group. The four groups of animals are injected into the gingival retraction part once every 7 days for 3 times, and are designed into a treatment course, and rats are sacrificed 7 days after the last treatment for whole blood collection and material drawing. And (3) carrying out hematological and biochemical detection on the fresh blood, observing the change of each index in the blood of four groups of animals, and testing the toxicity of graphene oxide to the animals. Observing the change condition of gingival tissue, freezing and storing gingival tissue for detecting the level of nucleic acid protein and comparing the therapeutic effect.
Various formulations were tested for biological safety in animals as shown in FIG. 12, +.i. in FIG. 12, for control, ■ for sPL formulation injection, go-sPL formulation injection, and T.sub.x for Go-sPL-CREKA formulation injection. The body weight of each group of rats increased during the experimental period, but there was no statistical significance (P > 0.05) in the body weight change of the rats in the three treatment groups between the groups compared to the control group. The effects of various injection formulations on the hematological and biochemical parameters of rats are shown in Table 2, and the whole blood and serum analysis parameters have no statistical significance compared with the control group, and the above two results indicate that Go and CREKA have no toxic effects on rats. The qRT-PCR method was used to detect COL-1, COL-3 and VEGF gene expression associated with soft tissue repair and regeneration, as shown in FIGS. 13-15, "+" indicates significant differences (P < 0.05) compared to the control group, "+", and "+" indicate significant differences (P < 0.01) compared to the control group (P < 0.001), and no sign indicates no statistical differences (P > 0.05) compared to the control group.
Compared with a control group, the three groups of treatment groups have up-regulated COL-1 and VEGF expression, and the difference is obvious, and the Go-sPL of COL-3 and Go-sPL-CREKA treatment groups have obviously up-regulated expression, so that the results show that the three groups of treatment groups have certain treatment effects on gingival retraction repair, and collagen and vascular regeneration can be promoted by increasing the expression of COL-1, COL-3 and VEGF, so that the growth of gingiva is promoted, wherein the Go-sPL-CREKA treatment effect is optimal.
TABLE 2 influence of injection formulations on rat hematology and biochemistry parameters
In Table 2, WBC is white blood cell count, RBC is red blood cell count, hgb is hemoglobin, hct is hematocrit, MCV is average red blood cell volume, MCH is average red blood cell hemoglobin, MCHC is average red blood cell hemoglobin concentration, PLT is platelet count, ALT is glutamic pyruvic transaminase, AST is aspartate aminotransferase, ALP is alkaline phosphatase, GGT is gamma-glutamyl transferase, DB is direct bilirubin, TB is total bilirubin, BUN is hematin nitrogen, crea is creatine, CPK is creatine phosphokinase, CK-MB is creatine kinase-MB, TP is total protein, TG is triglyceride, CHO is cholesterol, HDL is high density lipoprotein, LDL is low density lipoprotein.
Claims (6)
1. The sPL nano material for repairing gingival recession is characterized by being prepared from platelet lysate PL, exosomes, graphene oxide Go and fibrin targeting peptide CREKA, wherein the exosomes are extracted from dental pulp stem cells;
the graphene oxide Go is used as a nano carrier to wrap the sPL, and is modified by a fibrin targeting peptide CREKA to construct Go-sPL-CREKA;
The preparation method of the platelet lysate PL specifically comprises the following steps:
platelet enrichment is carried out, caCl 2 is added to the enriched platelets, the platelets are frozen at-80 ℃ for 8-10 h, then are thawed at 37 ℃ for 5-10 min, the repeated freezing and thawing are carried out for 10 times, the finally dissolved lysate is centrifuged at 6500rpm for 15-20 min, and the supernatant is reserved, thus obtaining platelet lysate PL.
2. The method for preparing the sPL nanomaterial for restoring gingival recession as set forth in claim 1, characterized in that the method includes the steps of:
Culturing the purified rat dental pulp stem cells to the third generation by using a complete culture medium, changing the culture medium to a corresponding culture medium without exosomes when the cells grow to 50% -60% by adherence, collecting culture supernatant when the cells grow to 90% -100% by adherence, and extracting exosomes;
step two, preparing platelet lysate PL, mixing PL with exosomes to obtain sPL;
Step three, mixing graphene oxide with the sPL prepared in the step two to obtain a Go-sPL suspension, then adding fibrin targeting peptide CREKA, and stirring for 2-3 hours to prepare a nanomaterial Go-sPL-CREKA;
the preparation method of the platelet lysate PL in the second step specifically comprises the following steps:
platelet enrichment is carried out, caCl 2 is added to the enriched platelets, the platelets are frozen at-80 ℃ for 8-10 h, then are thawed at 37 ℃ for 5-10 min, the repeated freezing and thawing are carried out for 10 times, the finally dissolved lysate is centrifuged at 6500rpm for 15-20 min, and the supernatant is reserved, thus obtaining platelet lysate PL.
3. The method for preparing the sPL nanomaterial for restoring gingival recession as set forth in claim 2, wherein the platelet enrichment method specifically includes:
The whole blood is centrifuged at 1500-2700rpm for 15-20 min to collect upper plasma, namely platelet-rich plasma, the platelet-rich plasma is centrifuged again at 3500-4300rpm for 20-25 min, the lower layer of the enriched platelets is left, and the volume of the enriched platelets is 1/10 of the volume of the whole blood.
4. The method of claim 2, wherein the ratio of PL to exosome is (40-60): 1.
5. A method for preparing a sPL nanomaterial for restoring gingival recession as claimed in claim 2 or 3, characterized in that the concentration of graphene oxide in the suspension of step three Go-sPL is 25-50 μg/mL.
6. The method of preparing a sPL nanomaterial for gingival recession restoration according to claim 5, wherein a volume ratio of the Go-sPL suspension to the CREKA peptide in the third step is 1 (2-5).
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