CN116693599A

CN116693599A - Method for preparing platelet-rich fibrin extract and method for culturing dental pulp stem cells by adopting PRFe

Info

Publication number: CN116693599A
Application number: CN202310700247.3A
Authority: CN
Inventors: 李莹; 张斌; 张文旋; 金晗
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2023-06-13
Filing date: 2023-06-13
Publication date: 2023-09-05

Abstract

A method for preparing platelet-rich fibrin extract (PRFe) and a method for culturing dental pulp stem cells by adopting PRFe relate to a method for preparing platelet-rich fibrin extract and a method for culturing Dental Pulp Stem Cells (DPSCs). The invention aims to solve the problem that the freeze thawing method has strong destructiveness to cell structures, and the dryness of DPSCs is gradually lost along with the increase of passage times in the in-vitro culture process. The method for preparing the platelet-rich fibrin extract comprises the following steps: ultrasonic treating at 40kHz for 15 min, standing at 4deg.C for 20 min, treating PRF for 3-11 times as one cycle, centrifuging, sucking supernatant, and filtering. Method for culturing dental pulp stem cells: isolation and culture of primary DPSCs, cell passage, cell cryopreservation, cell resuscitation, and three-dimensional culture of DPSCs. PRFe is prepared by a low-frequency cyclic ultrasonic method, and the PRFe and 3D culture are combined to be applied to the culture process of DPSCs, so that the proliferation, migration, colony formation and osteogenic differentiation capacity of DPSCs are successfully enhanced.

Description

Method for preparing platelet-rich fibrin extract and method for culturing dental pulp stem cells by adopting PRFe

Technical Field

The invention relates to a method for preparing platelet rich fibrin extract (PRFe) and a method for culturing Dental Pulp Stem Cells (DPSCs).

Background

Trauma, tumors, infections, surgical injuries, and the like, often create large area tissue defects. The regeneration of large-area tissue defects is difficult, the treatment method is limited, and the method is a serious difficulty in clinical treatment and is a research hot spot in the field of tissue engineering. In recent years, as stem cells have been intensively studied in the field of bone tissue engineering, dental pulp stem cells (Dental pulp stem cells, DPSCs) present in dental pulp tissue have become an important cell source for tissue defect repair. DPSCs are mesenchymal stem cells with self-renewal capacity and multidirectional differentiation potential, and have wide application prospects in tissue regeneration. However, in the in vitro culture process of DPSCs, the dryness of DPSCs gradually loses with the increase of passage times, and the multi-directional differentiation potential is also reduced. Therefore, it is particularly important to maintain the dryness of DPSCs during in vitro culture. The dryness of DPSCs is affected by various factors such as growth factors, immune regulation, culture modes, environment and the like, wherein the action of the growth factors and the change of the culture modes are two important factors affecting the dryness of DPSCs. In recent years, platelet rich fibrin (PRFe) extract is used in the fields of cell culture and regeneration of autologous tissues because of its rich growth factors and active proteins, but the preparation method of PRFe has not yet formed a systematic system.

The existing PRFe extraction method mainly comprises a freeze thawing method, and growth factor release is promoted and PRFe is obtained through repeated freeze thawing cycles. Research shows that the growth factor with certain concentration can promote cell proliferation and differentiation effectively when used in cell culture, and the growth factor with too high concentration can inhibit cell proliferation and differentiation. However, freeze thawing is more destructive to cellular structures, and research has also shown that repeated freeze thawing has a strong negative impact on growth factor content, and the addition of exogenous activators may limit PRFe application.

Disclosure of Invention

The invention aims to solve the technical problem that the freeze thawing method has strong destructiveness to cell structures, and the dryness of DPSCs is gradually lost along with the increase of passage times in the in-vitro culture process of DPSCs, and provides a method for preparing platelet-rich fibrin extract by a low-frequency circulating ultrasonic method.

The method for preparing the platelet rich fibrin extract comprises the following steps:

1. placing PRF obtained by centrifuging 10ml whole blood into a sterile 15ml centrifuge tube, and adding physiological sodium chloride solution to a volume of 10ml;

2. treating the PRF as a cycle by sonication at a frequency of 40kHz for 15 minutes and then standing at 4 ℃ for 20 minutes;

3. And (3) circularly treating the PRF for 3-11 times according to the operation sequence of the step two, centrifuging for 20 minutes at 5000 rpm, sucking the supernatant by using a pipettor, and filtering by using a sterile filter to obtain the platelet-rich fibrin extract.

And step three, circularly processing the PRF for 5 times according to the operation sequence of the step two.

And step three, circularly processing the PRF 7 times according to the operation sequence of the step two.

And step three, circularly processing the PRF 9 times according to the operation sequence of the step two.

The method for culturing dental pulp stem cells by using the platelet-rich fibrin extract comprises the following steps of:

1. isolation and culture of primary DPSCs

The method for extracting the primary dental pulp stem cells by enzyme digestion comprises the following specific steps:

(1) Placing the collected intact premolars which need to be removed due to orthodontic treatment in PBS buffer solution containing 10% of volume fraction of diab at 4 ℃ for marking;

(2) Removing periodontal ligament tissue on the surface of the tooth in an ultra clean bench, and repeatedly rinsing the tooth with PBS containing 10% of the volume fraction of the diab;

(3) Longitudinally dividing the tooth body by a sterile high-speed turbine mobile phone, splitting the tooth body when approaching a pulp cavity, taking out dental pulp tissue, removing root pulp, putting the dental pulp tissue into an alpha-MEM complete culture medium containing 10% of double antibodies by volume fraction, washing cleanly, shearing the dental pulp tissue, transferring the dental pulp tissue into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 revolutions per minute, sucking the supernatant, adding 1.5ml of type I collagenase with the concentration of 3mg/ml, and digesting in a water bath shaker at 37 ℃;

(4) When the tissue is digested to form transparent cloud, adding three volumes of alpha-MEM complete culture medium to stop digestion, centrifuging for 5 minutes at 1000 revolutions per minute, removing supernatant, re-suspending with the alpha-MEM complete culture medium, transferring the suspension into a T25 culture bottle, uniformly spreading at the bottom of the culture bottle, and placing into a cell incubator for primary culture;

(5) The cells are adhered to the wall for about 4 days, a culture bottle is not required to be moved during the period, and the cells are passaged when the cell quantity is fused to 80% -90%;

2. cell passage

After the cell amount is fused to 80% -90%, digestion and passage are carried out, and the specific steps are as follows:

(1) Pre-heating alpha-MEM complete culture medium and 1 XPBS to 37 ℃ in a water bath kettle in advance;

(2) The original medium in the T25 flask was aspirated off with a sterile Pasteur pipette and the cells were washed 2 times with 1 XPBS;

(3) Sucking off PBS, adding 1ml of pancreatin, and shaking the culture flask to make pancreatin fully contact with the cell surface;

(4) Cell shrinkage and rounding can be observed under an inverted microscope, the side wall of the culture bottle is gently knocked, and when most cells are in a suspension state, 2ml of complete culture medium is immediately added to stop digestion;

(5) Sucking the cell suspension by a sterile Pasteur pipette, repeatedly blowing the bottom of the culture bottle for 8-10 times, and transferring the cell suspension into a 15ml centrifuge tube;

(6) Centrifuging for 5 minutes at 750 rpm;

(7) The supernatant was aspirated off and 1ml of complete medium was added to resuspend the cells;

(8) Transferring the cell suspension into a T25 culture bottle with 2ml of complete culture medium added in advance, and shaking to uniformly inoculate cells at the bottom of the bottle;

(9) Placing the cell culture flask under culture conditions of 37deg.C and 5% CO ₂ Saturated wetIn a cell culture box at the degree, digestion and passage are carried out when the cell growth is fused to 80% -90%;

3. cell cryopreservation

After the cells grow and fuse to a passable state, the cells which are not used temporarily can be frozen for storage, and the steps are as follows:

(1) Putting the program cooling box into a refrigerator at 4 ℃ in advance for precooling;

(2) Taking dental pulp stem cells with the cell quantity of 80% -90%, washing once by 1 XPBS, adding 1ml of pancreatin for digestion, immediately adding 2ml of alpha-MEM complete culture medium to stop digestion when most cells are in a suspension state, and sucking cell suspension by a sterile Pasteur pipette for centrifugation;

(3) After centrifugation, the supernatant is sucked off, 1ml of frozen stock solution is added into a centrifuge tube to resuspend the cells, and then the cell suspension is transferred into a frozen stock tube;

(4) The freezing tube is placed into a pre-cooled program cooling box, and immediately transferred into a refrigerator at the temperature of minus 80 ℃, and then the freezing tube is transferred into liquid nitrogen;

4. Cell resuscitation

(1) The alpha-MEM complete culture medium is placed in a water bath kettle in advance and preheated to 37 ℃;

(2) A15 ml centrifuge tube was taken and 4ml of alpha-MEM complete medium preheated to 37℃was added thereto;

(3) Taking out the frozen cells in the liquid nitrogen, immediately placing the frozen tube into a water bath at 37 ℃, and rapidly shaking to melt the content in the tube;

(4) The outer wall of the mouth of the freezing storage pipe is wiped by 75% alcohol in the super clean bench;

(5) Opening the freezing tube, transferring the cell suspension in the freezing tube into a centrifuge tube with a culture medium added in advance, lightly blowing and uniformly mixing, and centrifuging for 5 minutes under the condition of 750 revolutions per minute;

(6) The supernatant was aspirated and 2ml of alpha-MEM complete medium was added to resuspend the cells;

(7) Transferring the cell suspension to a T25 culture bottle with 2ml of complete culture medium added in advance, and slightly shaking to uniformly inoculate cells at the bottom of the cell culture bottle;

(8) Placing the cell culture bottle in a cell culture box, replacing fresh complete culture medium after 24 hours, and performing digestion and passage when the cell growth is fused to 80% -90%;

5. three-dimensional culture of DPSCs

The polyacrylic acid mould is adjusted to be a circular mould with the diameter of 32mm, and the mould is perfused in a cell culture dish by agarose solution to construct a cell 3D culture system, and the specific steps are as follows:

(1) Soaking polyacrylic acid mould in 75% alcohol for 15 min, and taking out and placing in an ultra-clean bench for irradiation of ultraviolet rays for 2 hr;

(2) Preparing agarose solution with mass fraction of 1.5%, sterilizing at high temperature and high pressure, and placing in an oven for standby;

(3) Sucking 1.5ml agarose solution, placing in a 35mm cell culture dish, rapidly placing in a sterilized polyacrylic acid mould, and avoiding air bubbles as much as possible;

(4) Ventilating the super clean bench for 20 minutes, taking out the polyacrylic acid mould after agar is solidified, and obtaining a cell 3D culture system;

(5) Adding 1ml of alpha-MEM culture medium containing 1% volume fraction double antiserum and 6% volume fraction PRFe into a 3D culture system, placing the culture medium into a cell culture box, and inoculating the cells obtained in the step four onto the alpha-MEM culture medium for culturing for 72 hours to obtain dental pulp stem cell spheres;

(6) And inoculating the dental pulp stem cell spheres obtained by culturing in the 3D culture system into a T25 culture bottle, and recovering to 2D culture to obtain dental pulp stem cells.

The results of the invention show that the low-frequency ultrasound promotes the release of various active proteins and growth factors in the PRF, in particular the release of fibrinogen beta in the PRF. The PRFe prepared by the ultrasonic method and the PRFe prepared by standing for 24 hours are subjected to component analysis, the main components of the PRFe and the PRFe have no obvious difference, but compared with a standing group, the ultrasonic group partial protein and factors such as acid secretion protein rich in cysteine, spermatogenic protein, platelet factor 4 and the like have slightly low concentration, and the fibrinogen beta concentration is relatively high. The total time for circulating low-frequency ultrasonic to act on PRF and preparing PRFe is less than 6 hours, and the ultrasonic method is an efficient and feasible method from the aspect.

In the invention, DPSCs still have good cell activity after being cultured for 72 hours in 3D, which proves that the invention successfully constructs an agarose 3D culture system.

According to the invention, PRFe is prepared by a low-frequency cyclic ultrasonic method, and then PRFe and 3D culture are combined to be applied to the culture process of DPSCs, so that the proliferation, migration, colony formation and osteogenic differentiation capacity of DPSCs are successfully enhanced, and the expression level of dry markers NANOG and OCT4 in DPSCs is improved. The invention perfects the system for preparing PRFe by a low-frequency cyclic ultrasonic method, which is beneficial to the efficient preparation of PRFe and the application of PRFe in the fields of tissue engineering and stem cell research, and the autologous application of PRFe also avoids the occurrence of heterogeneous infection. The DPSCs obtained by applying PRFe to 3D culture has remarkable effect on maintaining the dryness, provides a new thought for the in-vitro culture mode of the DPSCs, and has important significance on the development of bone tissue engineering and regenerative medicine.

Drawings

FIG. 1 is a photograph of whole blood after centrifugation of the blood in experiment one;

FIG. 2 is a photograph of PRF gel in experiment one;

FIG. 3 is a photograph of PRFe prepared in experiment one;

FIG. 4 is a schematic illustration of PRFe preparation in experiment one;

FIG. 5 is a graph showing PDGF-AB concentration measurements at various cycle numbers in experiment one;

FIG. 6 is a graph of TGF-beta 1 concentration measurements at various cycle numbers in experiment one;

FIG. 7 is a statistical plot of PRFe component analysis and differential expression protein from ultrasonic PRF9 cycles in experiment one;

FIG. 8 is a statistical graph of PRFe composition analysis and differential expression protein prepared by standing PRF for 24 hours in experiment one;

FIG. 9 is a GO classification diagram of the differential protein in experiment one;

FIG. 10 is a cluster map of differential proteins in experiment one;

FIG. 11 is a volcanic plot of differential protein in experiment one;

FIG. 12 is a photograph of primary dental pulp stem cells from experiment one;

FIG. 13 is a photograph of passaged dental pulp stem cells in experiment one;

FIG. 14 is the results of flow cytometry detection of mesenchymal stem cell-specific markers CD73 of DPSCs in experiment one;

FIG. 15 is the results of flow cytometry detection of mesenchymal stem cell-specific markers CD90 for DPSCs in experiment one;

FIG. 16 is a flow cytometry detection of mesenchymal stem cell-specific markers CD105 of DPSCs in experiment one;

FIG. 17 is a flow cytometry detection of hematopoietic stem cell specific marker CD45 results from DPSCs in experiment one;

FIG. 18 is a flow cytometry detection of hematopoietic stem cell specific markers CD34 from DPSCs in experiment one;

Fig. 19 is a graph showing the effect of CCK8 assay on DPSCs proliferation of PRFe in experiment one, where P <0.05, P <0.01, P <0.001, t-test;

FIG. 20 is a photograph of a polyacrylic acid mold in experiment one;

FIG. 21 is a photograph of an agarose 3D culture system in experiment one;

FIG. 22 is a photograph of DPSCs cell spheres in 3D culture in experiment one;

FIG. 23 is a photograph of DPSCs cell spheres under a medium-high power microscope in experiment one;

FIG. 24 is a photograph showing the detection of cell activity of spheres of DPSCs by Calcein/PI fluorescent staining in experiment one;

FIG. 25 is a photograph showing the detection of cell activity of the spheres of DPSCs by Calcein/PI fluorescent staining in experiment one;

FIG. 26 is a photograph showing the detection of cell activity of spheres of DPSCs by Calcein/PI fluorescent staining in experiment one;

FIG. 27 is a photograph of the colony forming ability of a plate cloning experiment to detect DPSCs in different groupings in experiment one;

fig. 28 is a statistical plot of cell coverage in a well plate of experiment P <0.05, P <0.01, P <0.001, t-test;

FIG. 29 is a photograph of a scratch healing assay for DPSCs after incubation under different conditions in experiment one;

fig. 30 is a graph of cell mobility statistics in scratch healing experiments in experiment one, where P <0.05, P <0.01, P <0.001, t-test;

FIG. 31 is a statistical plot of the expression level of CDC42 mRNA in different groupings of DPSCs in experiment one;

FIG. 32 is a statistical plot of the expression levels of PCNA mRNA in different groupings of DPSCs in experiment one;

FIG. 33 is a statistical plot of the expression levels of RHOA mRNA in different groupings of DPSCs in experiment one;

FIG. 34 is a graph showing comparison of CDC42 and PCNA protein expression levels in different DPSCs groups in experiment one;

FIG. 35 is a graph showing comparison of the expression levels of RHOA protein in different DPSCs of group one experiment;

FIG. 36 is a graph of quantitative statistics of CDC42 protein expression levels in different DPSCs of group one experiment;

FIG. 37 is a graph showing the quantitative statistics of PCNA protein expression levels in different DPSCs in experiment one;

FIG. 38 is a graph of statistics of the quantification of RHOA protein expression levels in different groups of DPSCs in experiment one;

fig. 39 is an immunofluorescence assay to detect CDC42 expression in different sets of DPSCs in experiment one, P <0.05, P <0.01, P <0.001, t-test;

FIG. 40 is a graph showing the expression level of NANOG mRNA in DPSCs of different groups in experiment one;

FIG. 41 is a graph showing the expression level of OCT4mRNA in different groups of DPSCs in experiment one;

FIG. 42 is a graph showing the expression levels of NANOG protein and OCT4 protein in different DPSCs groups in experiment one;

FIG. 43 is a graph showing the quantitative statistics of NANOG protein expression levels in DPSCs of different groups in experiment one;

FIG. 44 is a graph of statistics of the quantification of the expression levels of OCT4 protein in different DPSCs in experiment one;

FIG. 45 is a photograph of alizarin red stain after 21d osteogenic differentiation of different DPSCs groups in experiment one;

fig. 46 is a semi-quantitative analysis of alizarin red staining results for different groups of DPSCs in experiment one with P <0.05, P <0.01, P <0.001, t-test.

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.

The first embodiment is as follows: the method for preparing the platelet-rich fibrin extract according to the present embodiment is as follows:

The second embodiment is as follows: the present embodiment differs from the first embodiment in that the PRF is cyclically processed 5 times in the third step in the operation sequence of the second step. The other is the same as in the first embodiment.

And a third specific embodiment: this embodiment differs from the first or second embodiment in that the PRF is cyclically processed 7 times in the third step in the operation sequence of the second step. The other embodiments are the same as those of the first or second embodiment.

The specific embodiment IV is as follows: this embodiment differs from one to three embodiments in that the PRF is cyclically processed 9 times in the third step in the operation sequence of the second step. The other is the same as in one of the first to third embodiments.

Fifth embodiment: the method for culturing dental pulp stem cells by using the platelet-rich fibrin extract according to the first embodiment comprises the following steps:

1. isolation and culture of primary DPSCs

2. cell passage

(6) Centrifuging for 5 minutes at 750 rpm;

(9) Placing the cell culture flask under culture conditions of 37deg.C and 5% CO ₂ In a saturated humidity cell incubator, carrying out digestion and passage when the cell grows and fuses to 80% -90%;

3. cell cryopreservation

4. Cell resuscitation

5. three-dimensional culture of DPSCs

The following experiments are adopted to verify the effect of the invention:

experiment one:

in the experiment, the method for preparing the PRFe by low-frequency cyclic ultrasound is perfected, clues are provided for the preparation and application of the PRFe in the future, and the components of the PRFe are analyzed. And then quantitatively applying PRFe to DPSCs culture under 2D and 3D, and respectively observing the influence of the combined actions of PRFe and 3D culture on DPSCs proliferation, migration, cloning, osteogenic differentiation capacity and dry marker expression, so that a better culture method is sought for maintaining the dryness of the DPSCs, and a foundation is laid for the application of the DPSCs in bone tissue engineering and regenerative medicine.

1. Materials and methods

1.1 Experimental materials

1.1.1 platelet rich fibrin

Healthy volunteers, 3 for each male and female, were recruited, aged between 23-35 years, and blood was collected and PRF was prepared. The experiment was approved by the ethical committee of the secondary hospital affiliated to the university of halbine medical science.

1.1.2 Experimental cells

The dental pulp stem cells used in the experiment are extracted from premolars which need to be extracted due to orthodontic treatment, the teeth are derived from oral maxillofacial surgery of a second hospital affiliated to the university of Harbin medical science, and all the processes are permitted by the ethical committee of the second hospital affiliated to the university of Harbin medical science.

1.1.3 Experimental primers

The primers involved in this experiment were designed by general Biotechnology. Verification was performed by NCBI on-line primer alignment tool (Nucleolide BlAST, https:// novopro. Cn/BlAST /). The relevant primer sequences are as follows:

1.2 Experimental major reagents

1.2.1 cell culture-related Agents

1.2.2 detection of growth factor content related Agents

1.2.3CCK-8 related experimental reagent

1.2.4real-time PCR-related Experimental reagents

1.2.5Western blot related experimental reagent

1.2.6 immunofluorescence-related Experimental reagents

1.2.7 antibodies for experiments

1.3 Experimental Main Equipment

1.4 Experimental methods

1.4.1PRFe preparation-related experiments

Preparation of 1.4.1.1PRF

Immediately after aspiration of 10 ml/tube of forearm venous blood of the volunteer using an additive-free vacuum blood collection tube, centrifugation was carried out at 3000 rpm for 10 minutes. After centrifugation, the blood sample in the blood collection tube can be seen to be divided into three layers: a red blood cell layer at the bottom, platelet rich fibrin in the middle and platelet poor plasma layer at the top. And (3) transferring the blood collection tube subjected to centrifugation into an ultra-clean bench, opening, slightly taking out the middle PRF gel layer by using sterile forceps, placing the PRF gel layer in a sterile culture dish, and shearing off the red blood cell layer at the bottom of the PRF gel layer by using the ophthalmology to obtain fresh PRF gel.

The experiment was approved by the ethical committee of the university of haerbin medical science, blood collection was performed by professionals and strictly following sterile principles, and volunteers informed consent and signed consent.

Extraction of related proteins in 1.4.1.2PRF

According to the PRF treatment mode, the treatment mode is divided into A, B, C, D, E, F six groups, and each group is divided into three repeated groups. The PRF can be treated by low-frequency ultrasonic to promote the release of the related protein, ultrasonic is carried out for 15 minutes, standing is carried out for 20 minutes at 4 ℃ as a cycle, and the related protein in the PRF can be better released by 3 times of cyclic treatment. The experiment increases the number of times of PRF cyclic treatment based on the earlier stage experiment so as to obtain the optimal cyclic number of PRF treated by low-frequency ultrasonic. In this experiment, group A was a group for 24 hours of standing, and group B, C, D, E, F was a group for low-frequency cyclic ultrasonic treatment.

Group A: PRF prepared by centrifugation of 10ml whole blood was placed in a sterile 15ml centrifuge tube and physiological sodium chloride solution was added to a volume of 10ml. After standing at 4℃for 24 hours, the mixture was centrifuged at 5000 rpm for 20 minutes. The supernatant was aspirated with a pipette, filtered through a sterile filter and transferred to a new 15ml centrifuge tube designated A1, A2, A3. Placing in a refrigerator at-80deg.C, and storing.

Group B: PRF prepared by centrifugation of 10ml whole blood was placed in a sterile 15ml centrifuge tube and physiological sodium chloride solution was added to a volume of 10ml. Ultrasonic treatment was carried out at 40kHz for 15 minutes, and standing at 4℃for 20 minutes as a cycle, and the cycle was carried out 3 times, followed by centrifugation at 5000 rpm for 20 minutes. The supernatant was aspirated by a pipette, filtered by a sterile filter, and transferred to a new 15ml centrifuge tube, designated B1, B2, B3. Placing in a refrigerator at-80deg.C, and storing.

C. D, E, F the times of ultrasonic treatment in four groups of low-frequency cycles are 5 times, 7 times, 9 times and 11 times (each time ultrasonic treatment is carried out at 40kHz for 15 minutes and standing is carried out at 4 ℃ for 20 minutes as one cycle), and the other treatment conditions are the same as those in the group B.

1.4.1.3ELISA method for detecting concentration of PDGF-AB and TGF-beta 1 growth factors in protein samples

ELISA kits and specimens were equilibrated at room temperature for 30 minutes before the start of the experiment, and standard substances, washing working solution, biotin-labeled antibody working solution, avidin-peroxidase complex (ABC) required for the experiment were prepared according to the requirements of the specification. According to the specification, the protein samples of the experiment were not diluted.

Sample activation: 60 μl of sample is taken, added with 30 μl of A solution, mixed well, incubated at room temperature for 10 minutes, then added with 30 μl of B solution, mixed well, and taken with 100 μl for subsequent sample detection.

The specific experimental steps are as follows:

(1) Adding a standard substance and a sample: and (3) setting a standard substance hole, a sample hole to be detected and a zero hole, respectively adding 100 mu l of standard substance or sample to be detected, adding 100 mu l of sample diluent into the zero hole, slightly shaking and uniformly mixing, and adding a sealing plate film.

(2) Incubating for 90 min at 37 deg.C by shaking table, taking ELISA plate, removing sealing plate membrane, removing liquid, and spin-drying without washing.

(3) The working solution of biotin-labeled antibody was diluted in advance at a ratio of 1:100 and equilibrated at 37℃for 30 minutes. Mu.l of biotin-labeled antibody working solution was added to each well, followed by incubation at 37℃for 60 minutes after sealing.

(4) The wells were discarded and dried, and each well was washed 3 times with 300 μl of 1 Xwash buffer.

(5) The working solution of horseradish peroxidase-labeled avidin was diluted in advance in a ratio of 1:100 and equilibrated at 37℃for 30 minutes. 100 μl of ABC working fluid was added to each well, the plates were closed and incubated at 37deg.C for 30 minutes.

(6) The wells were discarded and dried, and each well was washed 5 times with 300. Mu.l of 1 Xwashing buffer.

(7) Mu.l TMB color development solution is added to each well, the plates are closed, and incubated for 15-25 minutes at 37 ℃ in a dark place.

(8) 100 μl of stop solution was added to each well to stop the reaction, and the OD of each well at 450nm was measured by an ELISA reader.

(9) And (3) calculating results: and drawing a standard curve according to the standard substance concentration and the OD value obtained by measurement, and calculating the sample concentration.

1.4.1.4PRF analysis of related protein components

(1) The protein samples extracted from PRF were frozen in a refrigerator at-80℃for 3 hours.

(2) And (5) transferring the protein sample into a precooled freeze dryer for freeze drying overnight to obtain the freeze-dried PRF related protein.

(3) Proteomic detection analysis.

1.4.2 cell culture

1.4.2.1 isolation and cultivation of primary DPSCs

(1) The collected intact premolars, which are required to be removed due to orthodontic treatment, were placed in PBS buffer containing 10% of the volume fraction of the diabody at 4℃and marked and immediately sent to the laboratory.

(2) Periodontal ligament tissue was removed from the surface of the tooth in an ultra clean bench and the teeth were repeatedly rinsed with PBS containing 10% volume fraction of diab.

(3) The tooth is longitudinally divided by a sterile high-speed turbine mobile phone, when the tooth is close to a pulp cavity, the tooth is split, dental pulp tissue is taken out, after root pulp is removed, the tooth is put into an alpha-MEM complete culture medium containing 10% of double antibodies by volume fraction, the dental pulp tissue is washed clean, then the dental pulp tissue is sheared and moved into a 15ml centrifuge tube, centrifugation is carried out for 5 minutes at 1000 revolutions per minute, 1.5ml of type I collagenase with the concentration of 3mg/ml is added after supernatant is gently sucked, and digestion is carried out in a water bath shaker at 37 ℃.

(4) When the tissue is digested to form transparent cloud, adding three times of volume of complete culture medium to stop digestion, centrifuging for 5 min at 1000 rpm, removing supernatant, re-suspending with alpha-MEM complete culture medium, transferring the suspension into T25 culture flask, spreading at the bottom of the culture flask, and placing into cell incubator for primary culture.

(5) Cells will adhere to the wall for about 4 days, and the culture flask is not required to be moved during the period, and the cells can be passaged when the cell quantity reaches 80% -90%.

1.4.2.3 passage of cells

After the cell amount is fused to 80% -90%, digestion and passage are carried out, and the specific experimental steps are as follows:

(1) The alpha-MEM complete medium and 1 XPBS were pre-warmed to 37℃in a water bath.

(2) The original medium in the T25 flask was aspirated off with a sterile pasteur pipette and the cells were washed 2 times with 1 x PBS.

(3) PBS was removed, 1ml of pancreatin was added, and the flask was gently shaken to allow the pancreatin to fully contact the cell surface.

(4) Cell shrinkage rounding was observed under an inverted microscope, the flask side wall was gently tapped, and when most cells were in suspension, 2ml of complete medium was immediately added to terminate digestion.

(5) The cell suspension was aspirated by a sterile pasteur pipette, repeatedly blown down on the bottom of the flask several times, and then transferred to a 15ml centrifuge tube.

(6) Centrifuge at 750 rpm for 5 minutes.

(7) The supernatant was carefully aspirated off and 1ml of complete medium was added to resuspend the cells.

(8) The cell suspension was transferred to a T25 flask in which 2ml of complete medium had been previously added, and gently shaken to uniformly inoculate the cells at the bottom of the flask.

(9) Placing the cell culture flask under culture conditions of 37deg.C and 5% CO ₂ And (3) in a saturated humidity cell culture box, carrying out digestion and passage when the cells grow and fuse to 80% -90%.

1.4.2.4 cell cryopreservation

After the cells grow and fuse to a passable state, the cells which are not used temporarily can be frozen. The experimental procedure was as follows:

(1) And (5) putting the program cooling box into a refrigerator at 4 ℃ in advance for precooling.

(2) The cell digestion and centrifugation steps are described for passage of cells.

(3) After centrifugation, the supernatant was carefully aspirated, 1ml of the frozen stock solution was added to the centrifuge tube to resuspend the cells, and the cell suspension was transferred to a labeled frozen stock tube.

(4) And (3) placing the freezing tube into a pre-cooled program cooling box, immediately transferring the freezing tube into a refrigerator at the temperature of-80 ℃ and then transferring the freezing tube into liquid nitrogen.

1.4.2.5 cell resuscitation

(1) The alpha-MEM complete medium was preheated to 37℃in advance in a water bath.

(2) A15 ml centrifuge tube was taken and 4ml of alpha-MEM complete medium pre-heated to 37℃was added thereto.

(3) And taking out the frozen cells in the liquid nitrogen, immediately placing the frozen tube in a water bath at 37 ℃, rapidly shaking to enable the content in the tube to melt as soon as possible, and preventing water in the water bath from overflowing a tube cap in the shaking process so as to prevent cell pollution.

(4) The outer wall of the mouth of the freezing storage tube is wiped by 75% alcohol in the super clean bench.

(5) The freezing tube is opened, the cell suspension in the freezing tube is transferred into a centrifuge tube which is added with culture medium in advance, and the mixture is gently blown and mixed, and is centrifuged for 5 minutes under the condition of 750 revolutions per minute.

(6) The supernatant was carefully aspirated off and the cells were resuspended by adding 2ml of alpha-MEM complete medium.

(7) The cell suspension was transferred to a T25 flask in which 2ml of complete medium had been previously added, and gently shaken to uniformly inoculate cells at the bottom of the flask.

(8) The cell culture flask is placed in a cell culture box, after 24 hours, fresh complete culture medium is replaced, and digestion and passage are carried out when the cell growth is fused to 80% -90%.

1.4.2.6 flow cytometry detection of DPSCs surface markers

(1) The 3 rd generation DPSCs with good growth state were removed from the cell culture incubator and washed 2 times with 1 XPBS.

(2) DPSCs were digested and centrifuged following the step of cell passaging.

(3) After centrifugation, the supernatant was removed, and the cell pellet was obtained and washed 2 times with pre-chilled 1×pbs.

(4) Cells were resuspended using PBS containing 1% BSA and the cell density was adjusted to 1X 10 ⁶ And each ml.

(5) The desired antibody and 100. Mu.l of the cell suspension were added to the centrifuge tube and incubated at 4℃for 30 minutes in the absence of light.

(6) The tubes were centrifuged and the supernatant was aspirated, and washed twice with 1×pbs to remove unbound antibody.

(7) Cells were resuspended using cold PBS containing 1% bsa.

(8) Sealing with sealing film, and up-flowing cytometry.

1.4.3CCK-8 experiment

(1) Cells with good growth status of 3 rd to 4 th generation were removed from the incubator, digested and centrifuged (step co-passage).

(2) The digested cells were then subjected to a 3X 10 protocol ³ Density of individual/wells was seeded in 96-well plates.

(3) After the cell plating is finished, the cells are placed in an incubator for continuous culture.

(4) Cells were dosed 24 hours after plating, and different groups were tested 24 hours after dosing.

(5) Under dark conditions, 10. Mu.l of CCK-8 reagent was added to each well.

(6) The cells were placed in an incubator for further culture for about 4 hours.

(7) Absorbance (OD) values at 450nm were measured for each well using a microplate reader, and the results were saved and statistically analyzed.

1.4.4 three-dimensional culture of DPSCs

1.4.4.1 construction of three-dimensional culture System of DPSCs

The polyacrylic acid mould is adjusted to be a circular mould with the diameter of about 32mm, and the mould is perfused in a cell culture dish by agarose solution to construct a cell 3D culture system, and the specific steps are as follows:

(1) And (3) placing the polyacrylic acid mould in 75% alcohol for soaking for 15 minutes, taking out the mould after the soaking is finished, and placing the mould in an ultra-clean bench for ultraviolet irradiation for 2 hours.

(2) Preparing agarose solution with mass fraction of 1.5%, sterilizing at high temperature and high pressure, and placing in an oven for standby.

(3) 1.5ml of agarose solution was pipetted into a 35mm cell culture dish and carefully placed into the sterilized polyacrylic acid mold rapidly to avoid air bubbles as much as possible.

(4) And (3) ventilating the interior of the super clean bench for 20 minutes, and taking out the polyacrylic acid mould after agar is solidified to have a certain hardness to obtain the cell 3D culture system.

(5) 1ml of 1% double-antibody serum-free alpha-MEM culture medium is added into the 3D culture system, and the culture system is placed in a cell culture box for standby.

1.4.4.2 construction of DPSCs cell spheres

(1) The 3 rd generation dental pulp stem cells with good growth state are taken out from the cell incubator, washed, digested and centrifuged (step co-passage).

(2) The supernatant was removed, the pulp stem cells were resuspended in fresh medium, and cell counts were performed.

(3) Diluting the cell suspension to the desired density, inoculating 3X 10 cells into a 3D cell culture dish ⁵ And each.

(4) After 24 hours of culture, the cells were found to be tightly connected and aggregated with each other.

(5) After 72 hours of incubation, the cell spheres were collected for subsequent experiments.

1.4.4.3DPSCs culture Experimental groups

The experiment adopts different modes to culture DPSCs for 72 hours in the experimental process, and the specific experimental groups are as follows:

2D group (10% FBS, 1% double antibody alpha MEM medium, 2D culture).

2D culture+PRFe group (alpha-MEM medium containing 6% PRFe, 1% diabody, 2D culture).

3D group (10% FBS, 1% double antibody alpha MEM medium, 3D culture).

3D culture+PRFe group (alpha-MEM medium containing 6% PRFe, 1% diabody, 3D culture).

After 72 hours, each of the above groups of cells was restored to conventional 2D culture, and each group of DPSCs was examined for changes in clonotype, proliferation, migration, osteogenic differentiation capacity, and expression level of dry markers.

1.4.5real-time PCR experiments

Extraction of 1.4.5.1 cell RNA

(1) The cells were removed from the cell incubator, the culture broth was discarded, and 1ml of Trizol was added to each well, shaken well, and allowed to stand on ice for 10 minutes.

(2) The enzyme-free gun head sucks Trizol and repeatedly blows the hole bottom until the liquid is uniformly transparent.

(3) Trizol was transferred to 1.5ml enzyme-free EP tubes that were labeled in advance.

(4) Chloroform (0.2 ml) was added to each of the enzyme-free EP tubes, and the mixture was vigorously mixed for 15 seconds until the lysate became a pink milkshake, and the mixture was allowed to stand at room temperature for 3 minutes, and centrifuged at 12000 rpm at 4℃for 15 minutes.

(5) After centrifugation, the supernatant was transferred to a new 1.5ml enzyme-free EP tube, the same volume of isopropanol was added, and after gentle inversion and mixing, the mixture was allowed to stand at room temperature for 20 minutes.

(6) The mixture was centrifuged at 12000 rpm at 4℃for 10 minutes.

(7) The supernatant was discarded, 1ml of 75% ethanol was added to each tube, and the RNA pellet was gently rinsed.

(8) The mixture was centrifuged at 10600 rpm at 4℃for 5 minutes.

(9) The supernatant was discarded, the resulting RNA pellet was dried at room temperature, and DEPC water was added to dissolve the RNA pellet.

(10) And (3) measuring the A260/A280 ratio and the RNA concentration by using an ultra-micro ultraviolet spectrophotometer, and selecting an RNA sample with the A260/A280 ratio in the range of 1.8-2.0 for subsequent experiments.

1.4.5.2RNA reverse transcription

(1) Genomic DNA removal reaction:

(2) The reaction solution was placed in an enzyme-free eight-linked tube, and reacted in a PCR instrument. The reaction conditions were 42℃for 2 minutes, 4℃and C-cycle preservation.

(3) Reverse transcription reaction:

(4) The reverse transcription reagent is added into the octant tube of the step (2) and the reaction is carried out in a PCR instrument. The reaction conditions were 37℃for 15 minutes, 85℃for 5 seconds, 4℃and C were cyclically maintained.

1.4.5.3Real-time PCR

According toInstructions for premixexttm (DRR 047A) operate:

(1) The components and the amounts of the prepared PCR reaction liquid are as follows:

(2) Adding the prepared PCR reaction liquid into enzyme-free eight-linked tubes, setting three compound holes in each group, and reacting in an RT-PCR instrument:

pre-denaturation: 95℃for 10 min.

And (3) PCR reaction: the cycle was 95℃for 10 seconds, 60℃for 30 seconds, 72℃for 30 seconds, and 40 cycles were performed in total.

Dissolution profile: 95 ℃ for 10 seconds; 65 ℃ for 5 seconds; 95℃for 5 seconds.

(3) Analysis of results: the relative differences in gene expression in each group were analyzed using the 2-delta method with GAPDH as a housekeeping gene.

1.4.6Western Blot experiment

Preparation of 1.4.6.1 cell protein samples

(1) Preparing protein lysate: the volume ratio of RIPA and protease inhibitor is 99:1, and the lysate is prepared immediately.

(2) Cells were removed from the incubator, the original medium was discarded, and washed three times with 1 XPBS.

(3) 200 μl of the prepared protein lysate was added to each bottle of cells, and the mixture was left to stand on ice for 3 minutes.

(4) The whole cell at the bottom of the flask was scraped off with a cell scraper and transferred with a pipette into an EP tube marked in advance.

(5) The EP tube described above was placed on ice for 20 minutes during which time full vortexing was performed for 15-20 seconds every 5 minutes.

(6) Centrifugation was performed at a temperature of 4℃and a rotational speed of 12000 rpm for 20 minutes.

(7) The supernatant from the centrifugation was aspirated into a new EP tube and labeled. From this, 1. Mu.l was taken for detection of protein concentration.

(8) After the protein concentration determination was completed, the different samples were diluted to the same protein concentration.

(9) And adding a 5 XSDS-PAGE protein loading buffer with a sample volume of 1/4 to the protein sample, heating to 100 ℃ for 10 minutes, cooling and preserving at-20 ℃ for later use.

Determination of protein concentration by 1.4.6.2BCA method

(1) BSA protein standard was prepared at a concentration of 0.5mg/ml in advance.

(2) And preparing BCA working solution according to the number of protein samples to be tested.

(3) To the 96-well plate, 20, 19, 18, 16, 12, 8, 4, 0. Mu.l of a sodium chloride solution having a mass concentration of 0.9% was sequentially added, and then 0.5mg/ml BSA protein standard 0, 1, 2, 4, 8, 12, 16, 20. Mu.l was added to each well.

(4) 200 μl of BCA working solution was added to each of the wells, and the wells were allowed to react at 37℃for 30 minutes in the absence of light.

(5) The absorbance of each well at 560nm was measured by a microplate reader.

(6) A standard curve was drawn and the corresponding protein sample concentrations calculated.

1.4.6.3Western Blot (Western blot)

(1) The separation gel and the concentration gel were prepared according to the SDS-PAGE gel preparation.

(2) Electrophoresis: and respectively adding the protein Marker and the prepared protein sample into a protein loading hole, wherein the electrophoresis condition of the concentrated gel is 80V. And after the sample is electrophoresed to the separation gel, the sample is changed to 120V to continue electrophoresis.

(3) Transferring: the protein gel after electrophoresis was cut according to the molecular weight of the target protein, and a PVDF membrane having the same size as the gel was cut and activated with methanol. Placing filter paper, gel, PVDF membrane and foam cushion into a membrane transferring instrument according to sandwich interlayer, adding pre-cooled membrane transferring liquid, placing into an ice bag, and transferring for 0.5-2 hours under 300mA condition.

(4) After the transfer, the PVDF membrane was washed 3 times in TBST solution for 5 minutes each.

(5) Closing: the PVDF membrane was transferred to 5% skim milk and blocked for 60 minutes.

(6) Incubating primary antibodies: the primary antibody was prepared according to the antibody instructions, mixed well and incubated overnight at 4 degrees.

(7) The TBST was washed 3 times for 5 minutes each.

(8) Incubating a secondary antibody: secondary antibodies were prepared according to the antibody instructions and incubated for 90 min at room temperature.

(9) The TBST was washed 3 times for 5 minutes each.

(10) ECL developer develops, and the chemiluminescent imaging system images and photographs.

1.4.7 cell scratch healing experiments

(1) And drawing lines at the bottom of the six-hole plate by using a black marker pen, and marking.

(2) Taking out cells from the incubator, discarding the original culture medium, washing, digesting, centrifuging and counting cells.

(3) Inoculating cells into 6-well plates, 3X 10 cells per well ⁵ And each.

(4) When the cell polymerization degree reaches 100%, a 10 mu l sterile gun head is used for scratching the bottom of the six-hole plate, 3 scratches are scratched on each hole, and uniformity and consistency of scratches are ensured as much as possible.

(5) The detached cells were washed with 1 XPBS and photographed under an inverted microscope for recording.

(6) The cells were placed in an incubator for continuous culture for 24 hours.

(7) And photographing under an inverted microscope to record the healing condition of the scratches of the cells in different groups.

(8) Pictures of the same scratch field at 0 and 24 hours were bordered using ImageJ software, the scratch area was measured and plotted using GraphPadPrism8 analysis.

1.4.8 immunofluorescence staining experiment

1.4.8.1 preparation of cell climbing tablet

(1) The cell climbing sheet is cleaned, then is soaked in ethanol with the volume fraction of 75% for 30 minutes, and is then placed in an ultra-clean bench for airing, and is irradiated by ultraviolet rays for 1 hour.

(2) The processed climbing slices are placed in a 12-hole plate for standby.

(3) Taking out cells from the incubator, discarding the original culture medium, washing, digesting, centrifuging and counting cells.

(4) Seeding cells 3X 10 per well into 12 well plates ⁴ And each.

(5) After 24 hours, immunofluorescent staining was performed when cells grew to about 70% on the slide.

1.4.8.2 immunofluorescent staining

(1) The original medium was discarded and washed once with 1 XPBS in a 12-well plate with cells inoculated in advance.

(2) Fixing: the 4% paraformaldehyde fixative was fixed for 30 min, washed three times with 1 XPBS for 3 min each.

(3) Penetrating: cells were permeabilized on 0.3% Triton X-100 ice for 15 min, washed three times with 1 XPBS for 3 min each.

(4) Closing: the 3% BSA was blocked at room temperature for 1 hour.

(5) Incubating primary antibodies: the primary antibody was diluted according to the primary antibody instructions, and the preservative film was covered with 4℃and incubated overnight, and washed three times with 1 XPBS for 5 minutes each.

(6) Incubating a secondary antibody: the diluted fluorescent secondary antibody was added, incubated at room temperature for 90 minutes in the dark, the secondary antibody was pipetted off, and washed three times with 1 XPBS for 3 minutes each.

(7) Nuclear dyeing: hoechst33258 stained nuclei, incubated at room temperature for 5-8 min in dark place, washed three times with 1 XPBS for 3 min each.

(8) Sealing piece: a small amount of anti-fluorescence quenching agent is dripped on the glass slide, the cell climbing sheet is covered on the glass slide, and a small amount of neutral resin sealing sheet is dripped on the periphery for fixation.

(9) And photographing and observing under a fluorescence microscope.

4.4.9 Effect of three-dimensional culture and growth factors on DPSCs osteogenic differentiation

1.4.9.1 Induction of osteogenic differentiation by DPSCs

(1) Cells with good growth state of 3 rd generation were taken out from the incubator, digested and centrifuged (step co-passage).

(2) The digested cells were divided into four groups of cultures: 2D culture, 2D culture+PRFe, 3D culture, 3D culture+PRFe, and culturing for 72 hr.

(3) Digesting, centrifuging and counting the four groups of cells, and then obtaining the cell culture product according to the ratio of 10 multiplied by 10 ⁴ Density of each well was inoculated into six well plates, 1.5ml of alpha-MEM complete medium was added to each well, and the cells were placed in an incubator for culture.

(4) When the cell fusion degree reaches about 70%, the original culture medium is sucked and removed, and the culture medium is replaced by an osteogenesis induction culture medium for osteogenesis induction, and liquid is replaced every other day.

1.4.9.2 alizarin red staining

(1) DPSCs were removed and osteogenic induced for 21 days, medium was aspirated and washed once with 1 XPBS.

(2) 1.5ml of 4% paraformaldehyde fixing solution was added to each well, and the mixture was fixed at room temperature for 20 minutes.

(3) After the fixation was completed, the 1×pbs was washed three times for 3 minutes each.

(4) 1.5ml alizarin red staining solution was added to each well and stained at room temperature for 30 minutes.

(5) The alizarin red staining solution is absorbed, fully washed by distilled water, and then photographed and observed.

Semi-quantitative analysis of 1.4.9.3 alizarin red staining

(1) After alizarin red staining was completed, the well plate was rinsed three times for 5 minutes with 1×pbs.

(2) 1ml of 10% by mass cetylpyridinium chloride was added to each well and reacted for 20 minutes.

(3) The supernatant of the reaction solution was aspirated and added to a 96-well plate with 100. Mu.l each well, and three wells were set up for each group.

(4) The OD of each well was measured by a microplate reader at 560 nm.

(5) The supernatant was too dark and the dilution was followed by a reading.

1.4.10 plate clone formation experiments

1.4.10.1 cell plating

(3) The four groups of cells were digested, centrifuged, and counted, and inoculated into six well plates at a density of 300 cells/well, 1.5ml of alpha-MEM complete medium was added to each well, and the cells were placed in an incubator for continuous culture for 14 days.

1.4.10.2 crystal violet dyeing

(1) After the end of the clone formation, the medium was aspirated and washed 2 times for 3 minutes with 1 XPBS.

(2) 1.5ml of paraformaldehyde fixing solution with mass concentration of 4% was added to each well, and the mixture was fixed at room temperature for 30 minutes.

(4) 1.5ml of crystal violet staining solution was added to each well and stained for 30 minutes at room temperature.

(5) The crystal violet staining solution was aspirated, washed three times with 1×pbs for 3 minutes each, and then observed with photographing.

2 results

2.1PRF and PRFe preparation

After the collection of venous blood is centrifuged, three layers of vacuum blood collection tubes are visible: the top layer is light yellow anemia platelet plasma layer, the middle layer is PRF gel, and the bottom layer is erythrocyte layer (shown in figure 1). The PRF gel was removed from the blood collection tube and placed in a sterile petri dish, and the PRF gel was seen to be pale yellow jelly with a portion of red blood cells adhered to the lower end of the PRF gel (see FIG. 2). The bottom adhesion erythrocytes of PRF were removed using sterile scissors, and the resulting mixture was used to prepare PRFe as a clear pale yellow liquid after washing with sterile physiological saline (see FIG. 3). A schematic diagram of PRFe preparation is shown in FIG. 4.

2.2 determination of PDGF-AB and TGF-beta 1 concentrations in PRFe

The effect of sonication on the release of growth factors was determined by examining the levels of PDGF-AB and TGF- β1 in PRFe under different treatment conditions, and as a result, it was found that the release of both growth factors increased progressively with increasing number of sonication cycles and peaked at 9 cycles (FIGS. 5, 6), indicating that sonicating PRF was effective in promoting the release of growth factors.

2.3PRFe composition comparative analysis

In order to clearly determine the influence of specific components in PRFe and different preparation methods on the component composition, the PRFe (group E) prepared by ultrasonic PRF for 9 cycles and the PRFe (group A) prepared by PRF standing for 24 hours are subjected to component analysis and differential comparison. The results of the component analysis showed that the major components of the two groups of PRFe are similar (FIG. 7 and FIG. 8), the most specific weight of which is Albumin (ALB), and the other major components are Transferrin (TF), apolipoprotein A1 (APOA 1), immunoglobulin gamma-1 (IGHG 1), haptoglobin (HP), apolipoprotein B (ApolipoproteinB, APOB), which are important components in blood and are important for maintaining cell homeostasis.

Screening statistics (see fig. 9, 10, 11) of the differential proteins from the two groups of samples revealed that the concentration of various proteins in group E was lower than that in group a PRFe, including cysteine-rich acidic secreted protein (Secreted protein acidic and rich in cysteine, SPARC), protamine I (SemenogelinI, SEMG), platelet factor 4 (pf4), etc., but the concentration of Fibrinogen beta (FGB) was higher in group E than in group a. GO classification of differential proteins shows that the function of these differential proteins is mainly related to complement and coagulation cascade.

2.4 isolated culture of DPSCs

The extracted primary DPSCs can be seen as adherent cells around 1 week, the cells climb out in a divergent manner around dental pulp tissue fragments, the cells are long fusiform, uniform in morphology and good in growth state after passage (as shown in figures 12 and 13).

2.5 flow cytometry detection results of DPSCs

The surface markers of DPSCs were examined by flow cytometry, and the examination results showed positive expression of the mesenchymal stem cell-specific markers CD73 (98.7%), CD90 (97.8%) and CD105 (97.6%) and negative expression of the hematopoietic stem cell markers CD45 (0.2%) and CD34 (0.3%), as shown in FIGS. 14-18. The results indicate that the cells conform to the characteristics of mesenchymal-derived stem cells.

2.6 Effect of different concentrations of PRFe on DPSCs proliferation in vitro

The obtained PRFe with 9 cycles of ultrasonic PRF is used for replacing fetal bovine serum, and culture media containing different volume fractions of PRFe are prepared for DPSCs culture, and the influence of different concentrations of PRFe on DPSCs proliferation after 24 hours is observed. The results showed (FIG. 19) that the proliferation potency of DPSCs increased with increasing concentrations of growth factors and proteins in the medium within a certain range, and that DPSCs were most potent and most excellent in growth state when the volume fraction of PRFe contained in the medium was 6%. The difference between the groups was statistically significant (P < 0.01) compared to the group without PRFe, indicating that the proliferation of DPSCs was somewhat dependent on PRFe.

2.7 construction of agarose three-dimensional culture System

And constructing an agarose 3D culture system by using a polyacrylic acid mould, and culturing dental pulp stem cells in the 3D culture system for about 24 hours to form cell spheres. The cell spheres of dental pulp stem cells are uniformly distributed in a 3D culture system under observation of a microscope, the cell spheres are uniform in size and compact in structure, the surfaces of the cell spheres are smooth and complete under a living cell workstation, and the cell connection is tight (as shown in figures 20-23).

2.8 detection of Activity of cells in the spheroids of DPSCs cells

To examine the activity of cells in DPSCs cell spheres under 3D culture conditions, DPSCs cell spheres were stained using Calcein/PI double fluorescent staining (FIGS. 24-26). Most cells in the DPSCs cell sphere display green fluorescence, which indicates that most cells have good activity and state, and only a small number of cells die to display red fluorescence. The majority of dead cells accumulate in the center of the sphere, which may be caused by the lack of oxygen at the center of the sphere as the culture time increases and the cells expand.

2.9 Effect of different culture conditions on the clonality of DPSCs

In order to examine the change in the colony forming ability of DPSCs after culturing under different conditions, plate cloning experiments were performed, and the cell colonies were stained for crystal violet (FIG. 27, FIG. 28). Under the action of PRFe or 3D culture, the number of the formed clusters of DPSCs is increased, the number of the formed clusters of PRFe and 3D culture combined action is the largest, and the capability of forming the clusters of DPSCs is the strongest. In addition, DPSCs formed larger colony areas than 2D culture areas after 3D culture.

2.10 Effect of different culture conditions on DPSCs migration ability

To determine the effect of different culture conditions on the migration ability of DPSCs, the migration ability of DPSCs was examined by cell scratch healing experiments. The results show that the migration ability of DPSCs is slightly improved under the action of PRFe, but the migration ability of DPSCs can be more effectively enhanced by 3D culture, and the combined action effect of PRFe and 3D culture is most remarkable (shown in figures 29 and 30).

4.11 Effect of different culture conditions on CDC42, PCNA and RHOA expression

After confirming that both PRFe and 3D culture can improve the migration ability of DPSCs, the expression levels of proliferation cell nuclear antigen (Proliferating cell nuclear antigen, PCNA), cell division cyclin 42 (CDC 42) and RAS homologous gene family member A (Ras homolog gene family memberA, RHOA) in different groups of DPSCs were detected by Real-Time PCR and Western Blot experiments, and the expression of CDC42 was verified by immunofluorescence experiments. The protein plays an important role in regulating cell proliferation, adhesion and migration. The results show (FIGS. 31-39) that both PRFe and 3D cultures can enhance the expression of CDC42, PCNA and RHOA in DPSCs, wherein the effect of 3D culture on DPSCs is more remarkable, and the results are statistically significant.

2.12 Effect of different culture conditions on DPSCs drying Properties

To investigate whether different culture conditions have an effect on the dryness of DPSCs, mRNA and protein expression levels of the dryness markers NANOG and OCT4 in DPSCs were examined and the results are shown in FIGS. 40 to 44. 3D culture and PRFe have obvious promotion effects on NANOG and OCT4 expression in DPSCs, wherein 3D culture and PRFe have similar promotion effects on NANOG expression in DPSCs, and 3D culture effects are obviously better than those of PRFe on OCT4 expression in DPSCs. Overall, PRFe application in 3D culture promotes NANOG and OCT4 expression to the greatest extent, with optimal maintenance effect on the dryness of DPSCs.

2.13 Effect of different culture conditions on the osteogenic differentiation ability of DPSCs

After 21 days of DPSCs osteogenesis induction time, the effect of different culture conditions on DPSCs osteogenic differentiation ability was examined by alizarin red staining, and the results are shown in FIG. 45 and FIG. 46. Under the action of PRFe or 3D culture, the mineralized nodule formation number of DPSCs is increased, which suggests that the effect of PRFe or 3D culture can enhance the osteogenic differentiation capability of DPSCs. Wherein the 3D culture has stronger promotion effect on DPSCs osteogenic differentiation, and the combined application of the 3D culture and PRFe has the most obvious promotion effect on DPSCs osteogenic differentiation.

Discussion 3

DPSCs are one of the promising cell sources in tissue engineering, and have stronger cloning and multi-directional differentiation capabilities than BMSCs, but the capabilities are gradually lost with the increase of in vitro culture time. The level of DPSCs is affected by various factors such as gene expression, signal regulation, growth factor action, and microenvironment changes, wherein the application of growth factors and the change of culture modes are two important factors affecting the level of DPSCs. PRFe is rich in various growth factors and active proteins, and is easier to store and apply than PRF gel. However, at present, no systematic preparation system exists for PRFe, and PRFe plays an important role in the fields of autologous tissue regeneration and stem cell experiments, and autologous application of PRFe avoids the risks of heterologous infection and immune rejection and has a wide development prospect.

3.1PRFe preparation

The ultrasonic wave is a mechanical wave with extremely short wavelength and frequency more than 20KHz, the ultrasonic wave with frequency between 20KHz and 1MHz is low-frequency ultrasonic wave, and experiments show that cavitation and thermal effects generated by the low-frequency ultrasonic wave can promote aggregation and activation of blood platelets, so that the particles are removed to release growth factors. Furthermore, chen et al experiments have shown that platelets are strongly sensitive to mechanical forces, and that under the action of a mechanical signal, integrin αiibβ3 on the platelet surface enters an intermediate state between an active and an inactive state, which is functionally in a "mildly activated" state and can only be achieved by the transmission of a mechanical signal. More importantly, this intermediate state will transition to the active state under continued stimulation by the mechanical signal. Thus, during the ultrasonic preparation of PRFe, mechanical signal stimulation by the ultrasonic waves to platelets within the PRF will promote activation thereof through mechanical signal pathways on the platelet surface, thereby promoting the release of growth factors. The results of the invention show that the low-frequency ultrasound promotes the release of various active proteins and growth factors in the PRF, in particular the release of fibrinogen beta in the PRF. The PRFe prepared by the ultrasonic method and the PRFe prepared by standing for 24 hours are subjected to component analysis, the main components of the PRFe and the PRFe have no obvious difference, but compared with a standing group, the ultrasonic group partial protein and factors such as acid secretion protein rich in cysteine, spermatogenic protein, platelet factor 4 and the like have slightly low concentration, and the fibrinogen beta concentration is relatively high. The total time for circulating low-frequency ultrasonic to act on PRF and preparing PRFe is less than 6 hours, and the ultrasonic method is an efficient and feasible method from the aspect.

3.2 the volume fraction of PRFe in the culture medium is 6%, the DPSCs proliferation promoting effect is strongest

The experiment uses culture media containing different volume fractions (1%, 2%, 4%, 6%, 8% and 10%) of PRFe to culture DPSCs for 24 hours, and the cck8 method detection shows that the DPSCs have the strongest proliferation capacity when the volume fraction of PRFe is 6%. The above results indicate that in a certain concentration range, the proliferation capacity of cells increases with the increase of the content of the growth factors and the active proteins, but the addition of too high a concentration of the growth factors and the active proteins starts to inhibit the proliferation capacity of cells, which is similar to the experimental results of Uggeri et al. Thus, the volume fraction of PRFe applied to the medium was 6% in the subsequent experiments of this experiment. In addition, the promotion effect of PRFe on DPSCs proliferation is not only due to the inclusion of growth factors such as TGF- β1, PDGF, VEGF, and the like, but also due to the promotion effect of components such as fibrinogen and apolipoprotein in PRFe on cell proliferation.

3.3 construction of three-dimensional culture System and culture of DPSCs cell spheres

The 3D cell culture method is widely applied in the field of stem cell experiments, and compared with a culture system based on a bracket, the agarose bracket-free culture system selected in the experiment is simple and convenient to operate and low in cost. More importantly, the DPSCs cell spheres cultured by the culture system are easy to collect and are convenient to combine with materials in subsequent experiments and apply to the field of bone tissue regeneration. The surface of the constructed agarose 3D culture system is provided with a micropore structure, so that DPSCs can be uniformly distributed in the 3D culture system. In the 3D culture process of DPSCs, interaction between cells and extracellular matrixes of DPSCs cultured for about 24 hours can promote DPSCs to spontaneously aggregate to form DPSCs cell spheres, the structure of the DPSCs cell spheres is more compact along with the growth of culture time in the initial stage of 3D culture, and after the DPSCs cell spheres are cultured for 72 hours, fluorescent staining results show that the cells in the cell spheres have good survival states, but part of cells in the center of the cell spheres can undergo apoptosis due to hypoxia. Son et al used 3D cell culture dishes to culture DPSCs, and also found that DPSCs formed cell spheres after 24 hours without deformation for 72 hours. After cell spheroid formation, the level of pluripotency markers in DPSCs cell spheroids increases with increased adipogenic and osteogenic potential as compared to 2D cultured control cells. However, in the course of the experiment, it was found that in addition to the first 24 hours of cell aggregation, the cell proliferation capacity decreased, the cell cycle arrest and the apoptosis rate increased with the lapse of 3D culture time, and the oxygen consumption rate of living cells decreased with the lapse of polymerization time. In the experiment of the experiment, DPSCs still have good cell activity after being cultured for 72 hours in 3D, and the cell activity of the central part of the cell sphere gradually decreases after the culture time exceeds 72 hours. The reasons for the differences may be related to the difference of the 3D cell culture system and the difference of the culture medium components, and the invention also proves that the agarose 3D culture system is successfully constructed.

3.4PRFe and 3D culture to enhance the cloning, proliferation, migration and osteogenic differentiation ability of DPSCs

In this experiment, DPSCs were cultured for 72 hours under different culture conditions, and then transferred to a conventional 2D culture mode for culture, and the changes in cloning, proliferation, migration and osteogenic differentiation ability of DPSCs were examined. The result shows that PRFe and 3D culture have strong promotion effect on migration, proliferation and osteogenic differentiation of DPSCs, wherein the effect of 3D culture is superior to that of PRFe.3D culture can also significantly improve the clonality formation ability of DPSCs, while PRFe has no obvious effect. The effect of the combined application of PRFe and 3D culture is better than that of the single PRFe or 3D culture, which has reference significance for the in vitro culture of DPSCs. The result is consistent with a series of early experimental results, for example, the PRF extract is used for culturing human periodontal ligament cells in vitro, so that proliferation and osteogenic differentiation of the human periodontal ligament cells can be promoted, and the expression level of osteogenic related genes and transcription factors in the human periodontal ligament cells is up-regulated. The 3D culture can not only improve the cloning, proliferation, migration and osteogenic differentiation capacity of DPSCs, but also improve the proliferation capacity and activity of human chondrocytes in an in vitro culture environment. The experiment shows that PRFe and 3D cell culture play an important role in the proliferation, migration and osteogenic differentiation process of DPSCs.

Cell migration is a relatively complex biological process in which RHOA and CDC42 are important in regulating cell cycle, regulating cell morphology, RHOA plays a role in the assembly of stress fibers, CDC42 regulates filopodia formation, and thus affects the migration process of cells. The proliferation cell nuclear antigen PCNA is synthesized in the cell nucleus, and the expression level can be used as a reference index of the change of the proliferation capacity of the cell. Therefore, in this experiment, in addition to the migration ability of DPSCs detected by the cell scratch healing experiment, the expression levels of RHOA, CDC42 and PCNA in DPSCs after different culture conditions were detected, and the results again demonstrate the importance of PRFe and 3D cell culture in the cell proliferation and migration process.

3.5PRFe and 3D culture to increase expression level of DPSCs pluripotency markers

NANOG and OCT4 are important transcription factors affecting stem cell pluripotency, and play a role in stem cell renewal and pluripotency maintenance, and their expression levels are closely related to stem cell division, differentiation and stem cell potency. Compared with the traditional culture mode, the experiment shows that the 3D culture and PRFe effects can greatly improve the expression level of NANOG and OCT4 in DPSCs, the 3D culture effect is better than that of PRFe, and the result has statistical significance; the 3D culture combined with PRFe effect most significantly increased the expression levels of NANOG and OCT 4. In addition, growth factors or 3D cell culture to enhance stem cell pluripotency have also been reported in early experiments, such as Zhang et al, which uses a microgravity bioreactor to pellet adipose stem cells to maintain their dryness, short term alkaline fibroblast growth factor treatment enhanced OCT4, REX1 and NANOG mRNA expression levels and the ability of cell colony formation. However, in the experimental aspect of the influence of growth factors or active proteins on stem cell dryness, most of experimental objects are single growth factors or protein components, and the influence of PRFe on stem cell dryness is rarely reported. In the experiment, PRFe and 3D culture are jointly applied to the culture of DPSCs, and the proliferation, migration, colony formation, osteogenic differentiation capacity and drying potential of the DPSCs are obviously improved after the combined action of the PRFe and the 3D culture is found, so that the feasibility of the method is fully verified.

DPSCs are a type of mesenchymal stem cells with self-replication, self-renewal and multipotency, but excessive expansion of DPSCs in vitro results in reduced proliferation, migration and loss of stem potential. According to the invention, PRFe is prepared by a low-frequency cyclic ultrasonic method, and then PRFe and 3D culture are combined to be applied to the culture process of DPSCs, so that the proliferation, migration, colony formation and osteogenic differentiation capacity of DPSCs are successfully enhanced, and the expression level of dry markers NANOG and OCT4 in DPSCs is improved. The invention perfects the system for preparing PRFe by a low-frequency cyclic ultrasonic method, which is beneficial to the efficient preparation of PRFe and the application of PRFe in the fields of bone tissue engineering and stem cell experiments, and the autologous application of PRFe also avoids the occurrence of heterogeneous infection. The DPSCs obtained by applying PRFe to 3D culture has remarkable effect on maintaining the dryness, provides a new thought for the in-vitro culture mode of the DPSCs, and has important significance on the development of bone tissue engineering and regenerative medicine.

The experiment utilizes a low-frequency cyclic ultrasonic method to prepare PRFe, and compared with the condition that PRF stands for 24 hours to release growth factors and proteins, two indexes of TGF-beta 1 and PDGF-AB are selected for concentration detection, and a final method for preparing PRFe is established.

The effect of medium (1%, 2%, 4%, 6%, 8%, 10%) containing different volume fractions of PRFe on the growth status of DPSCs was examined by CCK-8 method, and the concentration of PRFe used for DPSCs culture was determined.

And constructing an agarose 3D culture system by using a polyacrylic acid mould, and detecting the DPSCs cell sphere activity under the 3D culture condition by using Calcein/PI fluorescent staining.

The effect of different culture conditions on the ability of DPSCs to form clonotypes was observed by crystal violet staining.

Cell scratch healing experiments observe the effect of different culture conditions on the migration capacity of DPSCs.

Real-Time PCR and Western Blot detect the expression of different cell proliferation nuclear antigens PCNA, cytoskeletal important regulating factors RHOA and CDC42 and cell stem property related indexes NANOG and OCT 4. Immunofluorescent staining detects the expression of the important regulator CDC42 of different groups of cytoskeleton. DPSCs under different culture conditions are subjected to osteoinduction for 21 days, and the osteogenic differentiation condition is detected by alizarin red staining.

Results: the low-frequency cyclic ultrasonic treatment PRF can efficiently extract the growth factors and related proteins in the PRF in a short time, and the content of the growth factors and related proteins in the PRFe increases along with the increase of the cyclic times of the low-frequency ultrasonic treatment PRF, and reaches a peak value at 9 cycles. The low frequency cyclic sonication increases the release of Fibrinogen beta (FGB) in PRF compared to 24 hours release in PRF, but the release of some components such as cysteine-rich acid secreted protein (Secreted protein acidic and rich in cysteine, SPARC), protamine I (semengelin I, SEMG 1), protamine II (SEMG 2), platelet factor 4 (pf4) is slightly lower than 24 hours release in PRF. PRFe prepared by a low-frequency cyclic ultrasonic method is selected for DPSCs culture, and when the volume fraction of PRFe in a culture medium is 6%, the DPSCs are best in growth state. The results of Calcein/PI fluorescent staining show that DPSCs cell spheres have good activity under 3D culture conditions. DPSCs showed stronger clonality formation, proliferation, migration, osteogenic differentiation capacity and dry potential (PCNA, RHOA, CDC, NANOG and OCT4 expression levels increased) than in 2D culture when PRFe or 3D culture was added. When PRFe and 3D cultures were combined on DPSCs, the expression levels of PCNA, RHOA, CDC, NANOG and OCT4 were further up-regulated.

Conclusion: perfecting the method for preparing PRFe by low-frequency cyclic ultrasound, and having strongest DPSCs proliferation capability when the volume fraction of PRFe in the culture medium is 6%. PRFe combines 3D culture, and compared with 3D culture or 2D culture added with PRFe, the method has stronger regulation and control effects on DPSCs clone formation, proliferation, migration, osteogenic differentiation and dryness.

Claims

1. The method for preparing the platelet-rich fibrin extract is characterized by comprising the following steps of:

3. and (3) circularly treating the PRF for 3-11 times according to the operation sequence of the step two, centrifuging for 20 minutes at 5000 rpm, sucking the supernatant by using a pipettor, and filtering by using a sterile filter to obtain the platelet-rich fibrin extract PRFe.

2. The method for preparing a platelet rich fibrin extract according to claim 1, wherein the PRF is cyclically treated 5 times in step three according to the operation sequence of step two.

3. The method for preparing a platelet rich fibrin extract according to claim 1, wherein the PRF is cyclically treated 7 times in step three according to the operation sequence of step two.

4. The method for preparing a platelet rich fibrin extract according to claim 1, wherein the PRF is cyclically treated 9 times in step three according to the operation sequence of step two.

5. A method of culturing dental pulp stem cells using the PRFe of claim 1, wherein the method of culturing dental pulp stem cells using the platelet rich fibrin extract comprises the steps of:

1. isolation and culture of primary DPSCs

2. cell passage

(6) Centrifuging for 5 minutes at 750 rpm;

3. cell cryopreservation

4. Cell resuscitation

5. three-dimensional culture of DPSCs