CN115105638B - Dental pulp-dentin complex regeneration promoting stent and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of biological materials. In particular to a bracket for promoting dental pulp-dentin complex regeneration and a preparation method and application thereof. The invention provides an deciduous tooth stem cell extracellular matrix scaffold (extracellular matrix scaffold of stem cells from exfoliated deciduous teeth, SHED-ECMS) for promoting regeneration of dental pulp-dentin complex and a preparation method thereof. The dental pulp bionic type SHED-ECMS is successfully prepared by firstly applying the stem cell extracellular matrix of the deciduous tooth, has dental pulp tissue morphology and structure, and can realize personalized preparation. The SHED-ECMS can provide a proper microenvironment for stem cell field planting, proliferation and differentiation which participate in dental pulp regeneration, can directionally induce the odontoblast of bone marrow mesenchymal stem cells to differentiate to form tubular prosthetic dentin and reticular dental pulp-like tissues so as to promote dental pulp-dentin complex regeneration and reduce irregular bone-like calcification formation in root canal after regenerative dental pulp treatment operation. The SHED-ECMS has excellent biocompatibility, no cytotoxicity, proper degradation performance and low immunogenicity, and is suitable for clinical application.

Description

Dental pulp-dentin complex regeneration promoting stent and preparation method and application thereof

Technical Field

The invention relates to the field of biological materials. In particular to a bracket for promoting dental pulp-dentin complex regeneration and a preparation method and application thereof.

Background

Pulp regeneration and continued root development are ideal targets for the treatment of young permanent tooth pulp and periapical disease. Regenerative dental pulp treatment (regenerative endodontic procedures, REPs) is a biologically-based treatment method that regenerates functional dental pulp tissue by inducing differentiation of stem cells introduced into the root canal, promoting continued development of dentin, pulp-dentin complex, root, etc., and becomes a new strategy for the treatment of young permanent dental pulp and periapical diseases. However, up to 62.1% of cases after REPs were found to have irregular calcification in the root canal (RAIC), even developing complete root canal occlusion, severely affecting pulp tissue regeneration and its physiological function. However, non-specific osteogenic differentiation of the bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMMSCs) recruited into the root canal is a major factor leading to RAIC.

Seed cells, scaffold materials, and growth factors are three major elements of tissue engineering techniques. Wherein the scaffold material and the growth factors together form a tissue regeneration microenvironment, which has important induction and regulation effects on seed cells, and the tissue regeneration curative effect is determined to a great extent. For REPs, blood clots formed by blood entering into root canal are scaffold materials for dental pulp regeneration, which can provide nutrition support for seed cells, but lack directional induced dentin differentiation, promote dental pulp-dentin complex microenvironment.

The development of biological scaffolds with the property of directionally inducing odontoblast differentiation is a new strategy for promoting the regeneration of pulp-dentin complex.

Disclosure of Invention

The deciduous tooth stem cells (stem cells from exfoliated deciduous teeth, SHED) are MSCs separated and extracted from the tooth pulp tissue of the deciduous tooth which is the only physiological deciduous organ of the human body, have stronger in-vitro expansion capacity, unique odontoblast differentiation, neurogenic differentiation and vascular regeneration promotion capacity, and more importantly, SHED expresses a large number of molecular signals related to tooth pulp development, participate in the tooth pulp development and regeneration process and play an important role; the extracellular matrix (extracellular matrix, ECM) is a complex grid structure constructed by proteins and polysaccharide macromolecular substances secreted by cells into extracellular mesenchyme, forms a highly organized extracellular microenvironment, and has important regulation effects on cell survival, tissue development and the like. The stem cell ECM scaffold obtained by the decellularization technology is considered as a novel natural biological scaffold material with great application prospect in the field of regenerative medicine.

The invention is to construct an dental pulp bionic type deciduous tooth stem cell extracellular matrix scaffold (extracellular matrix scaffold of stem cells from exfoliated deciduous teeth, SHED-ECMS), and characterize and identify biocompatibility, surface morphology, biological performance and the like; further, the effects of SHED-ECMS on BMMSCs adhesion, proliferation, migration, odontoblast differentiation and the like and the regeneration effect of dental pulp-dentin complex are researched through in vitro and in vivo experiments, and a foundation is laid for the application of SHED-ECMS as a novel natural biological scaffold material to REPs so as to reduce calcification and complications in root canal after REPs operation.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

The preparation method of the bionic SHED-ECMS for promoting the regeneration of the dental pulp-dentin complex is characterized by comprising the following steps of:

1) Inoculating the cultured cells to a cell plate after the cells grow to 80% confluence, replacing an extracellular matrix induction culture medium, continuously culturing for 14d, forming a white membrane-like substance at the bottom of a dish, and obtaining the cell membrane of the SHED when the edges are curled to indicate that the cell membrane is mature;

2) The surface of the SHED cell membrane is inoculated with SHED suspension, after the cells are attached, the cell membrane is wrapped in a mould, the mould is drawn out after 7d of culture, and the culture is continued for 7d, so as to obtain the dental pulp-like strip-shaped SHED cell aggregate (cell aggregate of stem cells from exfoliated deciduous teeth, SHED-CA).

3) SHED-CA was washed with PBS, decellularized with 3% Triton X-100 at 37deg.C for 72 h, and washed with PBS 3min X3 times to obtain dental pulp bionic SHED-ECMS.

The invention provides a dental pulp-dentin complex regeneration promoting functional product, which comprises the bracket, wherein the SHED-ECMS is derived from an extracellular matrix formed by the secretion of SHED.

The invention provides a dental pulp-dentin complex regeneration promoting functional product, which comprises the bracket, wherein the SHED is derived from a human physiological shedding organ, namely, a primary dental pulp tissue, is easy to obtain, has no iatrogenic trauma, has unique and superior stem cell performance, comprises stronger in-vitro expansion capacity, unique odontoblast differentiation, neurogenic differentiation and angiogenesis promoting regeneration capacity, and more importantly, expresses a large number of molecular signals related to dental pulp development, and plays an important role in dental pulp development and regeneration process.

The invention provides a dental pulp-dentin complex regeneration promoting functional product, which comprises the bracket, wherein the SHED-ECMS is prepared from extracellular matrixes secreted by dental stem cells, and can double-simulate dental pulp tissue morphology and bioactive components.

Furthermore, the SHED-ECMS bracket is prepared in a personalized way according to the tooth position and the size of the medullary cavity, and is derived from purely natural cell components.

The present invention provides the use of seed-ECMS in the preparation of a scaffold or material for promoting regeneration of pulp-dentin complex.

Furthermore, the SHED-ECMS has a loose porous homogeneous extracellular matrix structure, provides a space bracket for the growth of dental pulp regeneration seed cells, promotes the migration and homing of the seed cells into the root canal of the tooth, and the dental pulp regeneration seed cells comprise dental stem cells and jaw bone marrow stem cells.

Further, the SHED-ECMS is rich in bioactive components of molecular signals in early dental pulp development, can induce damaged dental pulp tissues to restart the breeding process, activate the stem property of dental stem cells, and promote the proliferation and differentiation of the stem cells to form dental pulp-dentin complex.

Further, the seed-ECMS is rich in odontoblasts characteristic proteins, including DSPP and DMP1, capable of directionally inducing differentiation of seed cells into odontoblasts to form pulp-dentin complex, reducing formation of irregular calcification in root canal.

Further, the SHED-ECMS is rich in bioactive components such as cytokines related to dental pulp regeneration, and provides an optimal and most approximate physiological microenvironment for dental pulp regeneration so as to promote dental pulp-dentin complex regeneration.

Compared with the prior art, the invention has the beneficial effects.

1) The self-assembly technology is adopted in the research, the SHED-ECM is firstly used for successfully preparing the dental pulp bionic SHED-ECMS, and the SHED-ECMS has excellent biocompatibility and no cytotoxicity and is suitable for clinical application.

2) The scaffold material can provide a proper microenvironment for stem cell field planting, proliferation and differentiation and promote dental pulp tissue regeneration.

3) The bracket material of the invention directionally induces the odontoblast of BMMSCs to differentiate to form tubular restorative dentin and reticular dental pulp-like tissues so as to promote the regeneration of dental pulp-dentin complex and reduce the formation of irregular bone-like calcifications in root canal after REPs operation.

Drawings

FIG. 1 is a map of the expression of SHED mesenchymal stem cell surface markers.

FIG. 2 is a graph showing the ability of SHED to differentiate into neurogenic.

FIG. 3 is a graph showing the ability of SHED to osteogenic differentiation.

Fig. 4 is a graph showing expression of mesenchymal stem cell surface markers from BMMSCs.

FIG. 5 is a graph showing the bone morphogenic differentiation ability of BMMSCs.

FIG. 6 is a graph showing the lipid-forming differentiation ability of BMMSCs.

FIG. 7 is a schematic diagram of the preparation of SHED-ECM scaffolds.

FIG. 8 is a SHED-ECMS construction diagram.

FIG. 9 is a view of the histological structure of SHED-ECMS.

FIG. 10 is a graph of the effect of DAPI staining to identify SHED-ECMS decellularization.

FIG. 11 is a diagram of DNA agarose gel electrophoresis and DNA quantification.

FIG. 12 is a SHED-ECMS microstructure observation.

FIG. 13 is a SHED-ECMS protein expression analysis chart.

FIG. 14 is a SHED-ECMS attachment chart of BMMSCs.

FIG. 15 is a graph of the proliferation capacity of BMMSCs measured by CCK-8.

FIG. 16 is a graph of the ability of Ki-67 immunofluorescence to detect BMMSCs proliferation.

Fig. 17 is a graph of flow cytometry detection of BMMSCs apoptosis levels.

Fig. 18 is a graph of BMMSCs migration ability for cell scratch assays.

FIG. 19 is a graph of the ability of a Transwell experiment to detect BMMSCs migration.

FIG. 20 is a graph of the mineralization ability of alizarin red S staining to detect BMMSCs.

FIG. 21 is a graph showing the Western blot analysis of the expression levels of bone formation/dentin associated proteins from BMMSCs.

Figure 22 is a schematic representation of a subcutaneous root segment implantation in the back of a mouse.

Fig. 23 is a histological view of pulp regeneration in a root segment.

FIG. 24 is a semi-quantitative analysis of the thickness of fresh dentin.

FIG. 25 is a semi-quantitative analysis of the number of odontoblasts.

FIG. 26 is a chart of immunofluorescence staining of odontoblasts.

FIG. 27 is a chart of immunohistochemical staining of odontoblasts.

Detailed Description

Example 1.

1. Primary separation culture and identification of SHED.

(1) Seed primary isolation culture.

The isolated teeth were placed in an alpha-MEM medium pre-chilled with 100U/mL penicillin/streptomycin. Repeatedly washing teeth with double-resistant PBS in an ultra-clean workbench, separating dental pulp tissue with sterile marrow extracting needle, fully cutting, adding 3 mg/mL type I collagenase and 4 mg/mL neutral protease, digesting for 30 min in a 37 ℃ incubator, swirling once every 10 min, adding an equal volume of SHED culture medium for neutralization, filtering with a 70 mu m cell sieve to obtain single cell suspension, inoculating in a culture dish with diameter of 10 cm, placing in a culture dish with diameter of 37 ℃ and 5% CO ₂ Culturing in an incubator. Culturing for 7d, half-volume liquid exchange, 10d, full-volume liquid exchange, and liquid exchange every 3d,when the cells grew to 90% confluence, the passaged cells were digested and 3-5 passages of cells were used for subsequent experiments.

(2) Flow cytometry detects stem cell surface markers of the seed.

Taking 3 rd generation SHED, centrifuging after pancreatin digestion, washing with PBS for 2 times, re-suspending cells with 0.5% BSA, counting, and adjusting cell density to 1×10 ⁶ Each tube was dispensed in FACS tubes at 100. Mu.L per tube. Centrifuging, discarding supernatant, respectively adding PE direct fluorescence labeled anti-human CD45, CD73, CD90 and CD105 antibodies, and FITC direct fluorescence labeled anti-human CD34 antibody 2 μl, taking IgG1 as isotype control, and incubating in ice in dark place for 1 h;1 mL of 0.5% BSA was neutralized, the supernatant was centrifuged off, and 2% paraformaldehyde (4% paraformaldehyde mixed with 0.5% BSA by equal volume) was added for fixation, and as seen in FIG. 1, the stem cell surface markers CD73, CD90 and CD105 were expressed by flow cytometry and the hematopoietic stem cell surface markers CD34, CD45 were expressed negatively.

(3) And (5) detecting the neurogenic differentiation capacity of the SHED.

Take the 3 rd generation SHED to 2X 10 ⁴ The cells are inoculated in 24-well plates, and after the cells are cultured until 70-80% confluence, the cells are replaced by nerve induction culture medium, and the liquid is replaced for 1 time every 2d, and the induction is carried out for 2 w.4% paraformaldehyde fixation, PBS wash 5 min X3 times, blocking buffer blocking 1 h (blocking buffer composition: 5% BSA/0.3% Triton X-100), beta III-Tubulin (1:1000) 4℃overnight incubation. PBS is washed for 5 min multiplied by 3 times, goat anti-rabbit fluorescent secondary antibody (1:200) 2 h is incubated at room temperature, a liquid sealing sheet containing DAPI anti-fluorescent quenching sealing sheet is subjected to observation and photographing by a fluorescent microscope, the observation can be carried out by figure 2, the in vitro nerve induction of SHED is 2w, and immunofluorescent staining shows that the expression of a nerve cell marker beta III-Tubulin is positive.

(4) And (5) detecting the osteogenic differentiation capacity of the SHED.

Take the 3 rd generation SEHD to 5 multiplied by 10 ⁴ Inoculating to 6-well plate at a density of 0-80% for cell culture, changing into osteoinductive medium, changing liquid 1 time every 2d, inducing 3w, observing mineralized nodule formation under mirror, washing with PBS for 5 min×3 times, fixing with 60% isopropanol for 1 min, adding 1 mL concentration 1% (1 g%mL) alizarin red S staining for 1 min, washing with deionized water to remove excess dye, observing and photographing under an inverted microscope, and observing from fig. 3, the in vitro osteogenesis/dentin induction of she 3w, which is seen as mineralized nodule formation, alizarin red S staining positive.

2. Primary culture and identification of BMMSCs.

(1) BMMSCs were cultured.

BMMSCs were purchased from ScienCell research laboratory, USA, and primary BMMSCs were inoculated into a 10 diameter cm petri dish and placed at 37℃in 5% CO ₂ Culturing in an incubator, changing liquid once every 3d, and digesting and passaging when the cells grow to 90% confluence, wherein 3-5-generation cells are used for subsequent experiments.

(2) Flow cytometry detects BMMSCs mesenchymal stem cell surface markers.

Taking 3 rd generation BMMSCs, centrifuging after pancreatin digestion, washing 2 times with PBS, re-suspending cells with 0.5% BSA, counting, and adjusting cell density to 1×10 ⁶ Each tube was dispensed in FACS tubes at 100. Mu.L per tube. Centrifuging, discarding supernatant, respectively adding PE direct fluorescence labeled anti-human CD45, CD73, CD90 and CD105 antibodies, and FITC direct fluorescence labeled anti-human CD34 antibody 2 μl, taking IgG1 as isotype control, and incubating in ice in dark place for 1 h;1 mL of 0.5% BSA was neutralized, the supernatant was centrifuged off, and 2% paraformaldehyde (4% paraformaldehyde mixed with 0.5% BSA by equal volume) was added for fixation and detection by flow cytometry. As shown in fig. 4, BMMSCs expressed mesenchymal stem cell surface markers CD73, CD90 and CD105, while hematopoietic stem cell surface markers CD34 and CD45 were expressed negatively.

(3) And detecting the osteogenic differentiation capacity of BMMSCs.

Taking 3 rd generation BMMSCs at a ratio of 5×10 ⁴ Inoculating the cells to a 6-hole plate at a density of 0-80%, culturing until the cells reach 70-80% confluence, replacing the cells with an osteoinductive medium, replacing the cells with the osteoinductive medium for 1 time every 2d, inducing 3w, observing mineralized nodules under a microscope, washing with PBS for 5 min multiplied by 3 times, fixing with 60% isopropanol for 1 min, adding alizarin red S with a concentration of 1% (1 g/mL) into each hole for 1 min, washing with deionized water to remove redundant dye, and observing and photographing under an inverted microscope. As shown in FIG. 5, BMMSCs bodies

External osteogenesis/dentin induction 3w, mineralized nodule formation, alizarin red S staining positive;

(4) And detecting the adipogenic differentiation capacity of BMMSCs.

Taking 3 rd generation BMMSCs at a ratio of 5×10 ⁴ Inoculating the cells to a 6-hole plate at a density of 0/mL, culturing the cells until the cells are 80% confluent, changing the cells to a lipid induction culture medium, changing the liquid for 1 time every 2d, inducing 4w, forming intracellular lipid drops under a mirror, washing the cells with PBS for 5 min multiplied by 3 times, fixing 4% paraformaldehyde for 30 min, washing the cells with PBS for 5 min multiplied by 3 times, dyeing the cells with oil red O for 30 min, washing the cells with deionized water to remove redundant dye, observing and photographing the cells under an inverted microscope, and inducing the cells to form lipid drops in vitro by BMMSCs for 4w, wherein the intracellular lipid drops are formed, and the oil red O is positive.

3. Preparation and characterization identification of SHED-ECMS.

(1) Preparation of SHED-ECMS.

As shown in FIG. 7, the 3 rd generation SEHD is taken at 5×10 ⁴ Inoculating the cells to a 6-hole plate at a density of one mL, changing extracellular matrix induction culture medium for 2-3 times per week after the cells grow to 80% confluence, continuously culturing for 14d, and obtaining the SHED membrane after the cells are mature when white membrane-like substances are formed at the bottom of a dish and the edges are curled. Seed cell patch surface seeding 1×10 ⁵ After cells are attached overnight, cell membranes are wrapped on a polytetrafluoroethylene tubular mold with the diameter of 0.8 mm, the polytetrafluoroethylene tubular mold is extracted after 7d of culture, and 7d of culture is continued, so that the strip-shaped SHED-CA is obtained. SHED-CA PBS is washed for 3min multiplied by 3 times, 3% Triton X-100 is decellularized for 72 h at 37 ℃, PBS is washed for 3min multiplied by 3 times, and the SHED-ECMS is prepared and stored in 1% double-antibody PBS solution for standby.

As shown in FIG. 8, SHED induction culture 14d, which is a net shape for cell multi-layer growth (FIG. 8A), was performed by inoculating SHED cells on the SHED membrane to obtain a SHED multi-layer membrane (FIG. 8B). The SHED cell membrane was folded and wound to form a strip-like pulp form, tightly textured SHED-CA (FIG. 8C), and decellularized to give a SHED-ECMS of about 13 mm length and about 1.5 mm diameter. The support is more transparent in color, soft and tough in texture, and has good operability (FIG. 8D)

(2) Characterization and identification of SHED-ECMS.

(1) And observing the histological structure of the SHED-ECMS.

SHED-ECMS was paraffin-embedded, serial sections of 5 μm, dewaxed with xylene, gradient ethanol water washed, hematoxylin stained for 3min, rinsed off flooding with deionized water, 1% hydrochloric acid-alcohol color separation, counterstaining with 0.5% eosin solution after 24 h% paraformaldehyde fixation. Gradient ethanol wash, xylene transparency, gum encapsulation, inverted microscope observation and photographing, H & E staining showed that the reed-ECMS did not see distinct nuclear structure, ECM was pink, homogeneous structure (a large number of bluish violet nuclear structures in non decellularized reed-CA), as shown in fig. 9, demonstrating that reed-ECMS effectively removed nuclear components while retaining ECM structure, reed-ECMS had a loose homogeneous extracellular matrix structure.

(2) DAPI staining identified the seed-ECMS decellularization effect.

SHED-ECMS sucrose gradient dehydrated, OCT embedded, 8 μm frozen sections after 4% paraformaldehyde fixation 24 h. Re-heating the frozen slices, and washing with PBS for 5 min multiplied by 3 times; the DAPI-containing anti-fluorescence quenching capper was observed under a fluorescence microscope and photographed, as shown in fig. 10, the seed-ECMS did not see the apparent DAPI positive staining nuclei, only a small amount of scattered DAPI positive staining nuclei material (no decellularization treatment seed-CA see the large amount of DAPI positive staining nuclei).

(3) And (3) identifying the seed-ECMS decellularization effect by a DNA agarose gel electrophoresis experiment and a DNA quantitative experiment.

DNA in the SHED-ECMS sample is extracted by using a DNA extraction kit, and SHED-CA is used as a control. Preparing agarose gel with the concentration of 1.5%, heating and melting for 1 min, cooling for a moment, dripping 2 mu L of fluorescent dye, uniformly mixing gel solution, pouring into an electrophoresis tank, solidifying at room temperature for 30-45 min in a dark place, and then placing into the electrophoresis tank. Adding the prepared DNA sample into the gel sample adding hole, switching on the power supply, 80-100V, ending electrophoresis when the electrophoresis position of the indicator reaches 1/2 of the electrophoresis position in the gel, and observing the electrophoresis belt and the position thereof by a gel imager. As shown in FIG. 11, agarose gel electrophoresis showed that the non-decellularized SHED-CA group showed a large number of DNA bands, whereas SHED-ECMS showed no obvious DNA bands, DNA standard solution was diluted to 1X TE buffer solution to 1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL in sequence and then added to 96-well plates, and samples were diluted in a ratio of 1:10 and then added to 96-well plates in a volume of 100. Mu.l per well; 100 μl of working solution is added to each well, incubated at room temperature in the dark for 5 min, and the absorbance of each well is detected by an ELISA OD 480 nm. As shown in FIG. 11, DNA quantitative analysis showed no significant DNA residues (P < 0.001) in the SHED-ECMS group.

(4) And observing the microstructure of the SHED-ECMS.

SHED-ECMS was fixed overnight at 2.5% glutaraldehyde and washed 10 min X3 times with PBS; sequentially dehydrating with 30%, 50%, 70%, 90% and 100% ethanol gradient, and dehydrating for 15 min at each concentration; and (3) freeze-drying treatment 2 h, metal spraying and film plating treatment, and observing the surface structure of the material in a scanning electron microscope. As shown in FIG. 12, the scanning electron microscope showed that the SHED-CA fibers were closely arranged in a rope shape, the structure of the SHED-ECMS was not significantly changed compared with the structure of the SHED-CA fibers, but the fiber arrangement was more porous, and the result shows that the SHED-ECMS effectively removes the nuclear component while retaining the ECM structure, and the SHED-ECMS has a loose and homogeneous extracellular matrix structure.

(5) And SHED-ECMS protein expression analysis.

SHED-ECMS sucrose gradient dehydrated, OCT embedded, 8 μm frozen sections after 4% paraformaldehyde fixation 24 h. Frozen sections were rewashed, washed 5 min X3 times with PBS, blocked with blocking buffer 1 h (blocking buffer composition: 5% BSA/0.3% Triton X-100), anti-fibreonectin (1:250), anti-collagenI (1:250), anti-DMP1 (1:250), anti-DSPP (1:250), incubated overnight at 4 ℃. PBS washes 5 min×3 times, room temperature incubates goat anti-rabbit fluorescent secondary antibody (1:200) 2 h, DAPI anti-fluorescent quenching sealing sheet-containing liquid sealing sheet, fluorescent microscope observations and photographs, by FIG. 13 shows that SHED-ECMS positive expressed extracellular matrix markers, fibroectin, collagen I (FIG. 13A), while positive expressed dentin-associated proteins DMP1 and DSPP (FIG. 13B), compared to SHED-CA, SHED-ECMS group did not see DAPI positive nuclei (FIGS. 13A, B); the above results demonstrate that seed-ECMS is effective in retaining extracellular matrix components and is rich in dentin-associated proteins.

4. SHED-ECMS effect on BMMSCs.

(1) BMMSCs attach at SHED-ECMS.

BMMSCs at 5X 10 ⁴ Inoculating individual cells/mL to SHED-ECMS, placing in a 5% CO2 incubator at 37 ℃ for culturing 24 h, and taking normal culture BMMSCs as a control group; fixing in 4% paraformaldehyde for 30 min, washing with PBS for 5 min×3 times, and activating Green at room temperature ^TM 488. Incubating for 30 min; PBS is washed for 5 min multiplied by 3 times, the anti-fluorescence quenching sealing piece liquid sealing piece containing DAPI is used for observing the expression condition of the cytoskeleton F-actin by a fluorescence microscope, and as shown in figure 14, F-actin immunofluorescence staining shows that BMMSCs are well attached and grown on the surface of the SHED-ECMS, and compared with the BMMSCs which are normally cultured, the BMMSCs are more obvious in cell expansion on the surface of the SHED-ECMS, are in a long fusiform shape and are regularly arranged along the ECM fiber structure.

(2) Preparation of SHED-ECMS conditioned Medium.

According to International Standard ISO10993-12, SHED-ECMS extract was extracted according to standard of 0.1 g/mL, namely 1 mL alpha-MEM culture medium was added to each 0.1g rack, the mixture was subjected to leaching at 37℃for 72 h, and centrifuged at 1500 rpm for 5 min to obtain supernatant, and the supernatant was filtered through a 0.22 μm filter to prepare a extract. The penicillin/streptomycin diabodies were prepared as SHED-ECMS conditioned medium in 15% fetal bovine serum, 100U/ml penicillin/streptomycin diabodies.

(3) Influence of SHED-ECMS on BMMSCs cell Activity.

(1) The proliferation capacity of BMMSCs is detected by a CCK-8 method.

BMMSCs were inoculated at 3,000 cells/well into a culture plate (96 well plate), and SHED-ECMS conditioned medium was added and placed at 37℃in 5% CO ₂ Culturing in an incubator, and taking normal culture BMMSCs as a control group; after 1 d, 3d, 5 d and 7d, 10 μl of CCK8 solution was added to each well, the wells were incubated in an incubator in the dark for 2 h, and the absorbance of each well was measured by an ELISA detector OD 450nm, as shown in FIG. 15, the CCK-8 results showed no significant difference in cell proliferation rates (P > 0.05) of the SHED-ECMS BMMSCs at 1 d, 3d, 5 d and 7d compared to the normal BMMSCs control group, indicating that SHED-ECMS had no significant effect on BMMSCs activity.

(2) And detecting the proliferation capacity of BMMSCs by a Ki-67 immunofluorescence method.

BMMSCs at 2X 10 ⁴ Inoculating the individual cells/holes to a culture plate (24-hole plate) with preset cell climbing plates, adding SHED-ECMS condition culture medium, placing in a 5% CO2 incubator at 37 ℃ for culturing 48 h, and taking normal culture BMMSCs as a control group; 4% paraformaldehyde fixed at room temperature, PBS wash 5 min X3 times, blocking buffer blocking 1 h (blocking buffer composition: 5% BSA/0.3% Triton X-100), anti-Ki-67 (1:800) incubated overnight at 4deg.C. The goat anti-rabbit fluorescent secondary antibody (1:200) 2 h was incubated at room temperature after 5 min X3 times in PBS, and the liquid seal with DAPI anti-fluorescent quenching seal was observed with a fluorescent microscope and photographed. The Ki-67 positive cell number and total cell number were counted and the Ki-67 positive cell percentage was calculated, as shown in FIG. 16, and Ki-67 immunofluorescence staining showed that the ratio of Ki-67 positive BMMSCs cells of the SHED-ECMS group was 67% and that there was no statistical difference (P > 0.05) compared to the control group, indicating that SHED-ECMS had no significant effect on BMMSCs activity.

(3) Flow cytometry detects the level of apoptosis of BMMSCs.

BMMSCs at 5X 10 ⁵ Inoculating the cells/holes to a culture plate (6-hole plate), adding SHED-ECMS condition culture medium, placing in a 37 ℃ and 5% CO2 incubator for culturing, and taking normal culture BMMSCs as a control group; cell digestions were centrifuged at 1 d, 3d, 5 d and 7d, PBS resuspended and 1×105 BMMSCs were added to 100. Mu.L Binding buffer to resuspended cells, 1. Mu.L Annexin V-APC was added, incubated at room temperature for 10 min, 1. Mu.L 7AAD was added, incubated at room temperature for 5 min, and flow cytometry detected the Annexin V/7AAD double positive cell ratio. As shown in FIG. 17, the apoptosis rate of BMMSCs increased slightly with the in vitro culture time, both in the control group and in the SHED-ECMS group, but BMMSCs were apoptotic at 1 d, 3d, 5 d and 7d, early stage (Annexin V ⁺ /7AAD ^- ) Late apoptosis (Annexin V) ⁺ /7AAD ⁺ ) Cell necrosis (Annexin V) ^- /7AAD ⁺ ) No significant difference in cell ratio (P > 0.05) indicates that SHED-ECMS has no significant effect on BMMSCs activity.

(4) Influence of SHED-ECMS on the migration ability of BMMSCs.

(1) Cell scratch experiments test BMMSCs migration capacity.

BMMSCs at 5X 10 ⁴ Inoculating at a density of 5% CO at 37deg.C in 6-well plate ₂ Culturing in an incubator; after the cells grow to 90% confluence, a 200 mu L sterile gun head makes cell scratches along the largest diameter of the pore plate, free cell fragments are removed by gently flushing with PBS, a SHED-ECMS conditioned medium is added, BMMSCs are used as a control group for normal culture, and the cells are observed and photographed under an inverted microscope of 0 h, 6 h, 12 h and 18 h after the scratches. Analysis of cell scratch healing Rate Using Image J software, FIG. 18 shows that the healing Rate of SHED-ECMS BMMSCs cell scratches was significantly improved compared to the control group (P<0.001 The SHED-ECMS is suggested to significantly enhance the migration ability of BMMSCs.

(2) And a Transwell experiment is used for detecting the migration capacity of BMMSCs.

Using an 8 μm pore size Transwell cell system at 1X 10 ⁵ Density of individuals/wells inoculated into Transwell cells; adding SHED-ECMS conditioned medium outside the chamber, standing at 37deg.C and 5% CO ₂ Culturing 24 h in an incubator, and taking normal culture BMMSCs as a control group; the Transwell cells were removed, 4% paraformaldehyde fixed at room temperature, 0.5% crystal violet stained, rinsed with PBS, and photographed under an inverted microscope. Analysis of the number of crystal violet positive cells under the Transwell chamber using Image J software, fig. 19 shows that Transwell cell migration experiments show that the number of BMMSCs cells migrated through the Transwell chamber by the seed-ECMS group is significantly increased compared to the control group, which suggests that seed-ECMS improves BMMSCs migration capacity (P< 0.001）。

(5) Effect of speed-ECMS on the osteogenic/odontogenic differentiation capacity of BMMSCs.

(1) Bone formation/dentin induction culture of BMMSCs.

BMMSCs at 5X 10 ⁴ Inoculating at a density of one mL into 6-well plate, adding SHED-ECMS conditioned medium, standing at 37deg.C in 5% CO ₂ Culturing 3d in an incubator, and taking normal culture BMMSCs as a control group; the osteogenesis/odontogenesis induction medium was changed, every 3d exchanges of fluid, the induction culture 10d collected proteins, and the induction culture 3w was subjected to mineralization nodule staining.

(2) Alizarin red S staining detects BMMSCs mineralization capacity.

Osteogenesis/odontogenesis is induced to culture for 3w, fixed for 1 min with 60% isopropanol, washed for 5 min×3 times with PBS, stained with 1 mL concentration 1% (1 g/mL) alizarin red S for 1 min per well, rinsed with deionized water to remove excess dye, and photographed under an inverted microscope. Analysis of alizarin red S positive staining area percentage using Image J software, fig. 20 shows that alizarin red S staining shows that in vitro osteogenesis/odontogenesis induced 3w, both reed-ECMS and control groups see mineralized nodule formation, but the number of mineralized nodules is significantly increased (P < 0.001) in the reed-ECMS group compared to the control group, indicating that reed-ECMS can increase BMMSCs mineralization capacity.

(3) Western blot detects the expression level of BMMSCs osteogenesis/odontogenesis-related proteins.

The osteogenesis/odontogenesis is induced to culture for 10d, the pre-cooled PBS is used for washing, 60 mu L/hole of cell lysate is added, cells are scraped and collected, the cells are cracked on ice for 1 h, vortex is carried out once every 15 min, centrifugation is carried out at 12,000 rpm for 5 min at 4 ℃, supernatant is extracted, and the supernatant is stored at-80 ℃ for standby. The BCA kit measures protein concentration and protein denaturation at 100 ℃ for 8 min. 20. Mu.g protein samples were subjected to Tris-Glycine gel electrophoresis (electrophoresis conditions: 125V, 1.5 h), PVDF membrane transfer (membrane transfer conditions: 30V, 2 h), and then subjected to shaking table blocking at room temperature of 1:1 h, anti-ALP (1:200), anti-RunX2 (1:1000), anti-DSPP (1:1000), anti-DMP1 (1:1000), and overnight at 4 ℃. TBST was washed 3 times and anti-rabbit or anti-mouse fluorescent secondary antibody 1 h was incubated on a room temperature shaking table. The infrared fluorescence scanning imaging system detects protein bands. Image pro plus 6.0 software was used to analyze the strips. As shown in fig. 21, western blot detection shows that in vitro osteogenesis/odontogenesis induced culture is 10d, compared with the control group, the expression levels of dentin-associated protein DSPP and DMP1 in the SHED-ECMS BMMSCs are significantly increased (P < 0.001), while the expression level of bone-associated protein ALP and RunX2 is not significantly changed (P > 0.05), which indicates that the SHED-ECMS can directionally induce the odontogenesis induced differentiation of the BMMSCs.

(6) Animal experiment study of SHED-ECMS to promote pulp regeneration.

(1) And (5) establishing a nude mouse back subcutaneous root segment transplanting model.

Healthy premolars that need to be extracted due to orthodontic are collected. Transversely cutting at 1/3 of the root neck to prepare a 5 mm Gao Yagen segment, and removing cementum, dental pulp tissues and prophase dentin; 17% EDTA (ethylene diamine tetraacetic acid) is treated for 5 min at room temperature, deionized water is subjected to ultrasonic oscillation for 10 min, and the operation is repeated twice; after high temperature sterilization, the root segments were stored in PBS containing 1% of diabody, at 4℃for use. The experiments were divided into two groups: (1) experimental group: SHED-ECMS stent group: SHED-ECMS composite 1×10 ⁶ Individual BMMSCs transplants, (2) control group: gelatin sponge group: gelatin sponge composite 1X 10 ⁶ Transplanting individual BMMSCs; as shown in FIG. 22, 0.2ml of 1% pentobarbital sodium was used for the abdominal anesthesia of mice, alcohol cotton balls were used for sterilizing the skin of the experimental area on the back of the nude mice, a longitudinal incision of about 1cm was made in the middle of the back of the nude mice, root fragments were blunt-separated from the implantation space on both sides of the back using ophthalmic forceps, root fragments containing different contents (see experimental group for details) were implanted into the subcutaneous parts of the back of the mice, the incision was sutured, and specimens were collected for 8 weeks.

(2) Histological staining.

The root section is obtained by taking materials, fixing 24H by 4% paraformaldehyde, dehydrating by gradient ethanol, embedding by paraffin, preparing tissue slices with the thickness of 5 mu m, staining by H & E, and photographing by observation by an optical microscope. 3 views of each specimen were taken, and Image J software measured the number of odontoblasts and dentin regeneration thickness in a single view. Immunofluorescence and immunohistological staining observed DSPP expression. H & E staining shows that tooth pulp-like tissue is formed in root canal of tooth root segment of SHED-ECMS group, a large number of dental pulp cells are interlaced to form net shape, distributed neovascular formation is scattered, the inner wall of root canal is formed by continuous neotubular dentin, the inner side of dentin is arranged by high columnar odontoblast-like cell polarity, and cell protuberance is deep into dentinal tubule to form dental pulp-dentin complex structure; however, the control group of the newly-grown dental pulp-like tissues were loosely arranged, were organized in a random manner, and were formed with a small amount of new blood vessels, and no obvious newly-grown dentin layer and odontoblast formation were seen (fig. 23). Semi-quantitative analysis showed a significant increase in both neodentin thickness and number of odontoblasts in the reed-ECMS group compared to the control group (P < 0.001) (fig. 24, 25). DSPP is a secreted protein, mainly expressed in cytoplasm and extracellular matrix, and immunofluorescence and immunohistochemical staining results showed that SHED-ECMS group was highly expressed as dentin marker protein DSPP, which was significantly different from control group (FIGS. 26, 27). The results indicate that SHED-ECMS is effective in promoting dental pulp-dentin complex regeneration.

Claims (9)

1. A method for preparing an pulp bionic type deciduous tooth stem cell extracellular matrix scaffold (extracellular matrix scaffold of stem cells from exfoliated deciduous teeth, speed-ECMS) for promoting the regeneration of a pulp-dentin complex, which is characterized by comprising the following steps of:

1) Culturing the cultured deciduous tooth stem cells (stem cells from exfoliated deciduous teeth, seed), inoculating to a cell plate, changing extracellular matrix induction medium when the cells grow to 80% confluence, continuously culturing for 14d, forming a white film-like substance at the bottom of a dish, and obtaining the seed cell membrane when the edge is curled to indicate that the cell membrane is mature;

2) Inoculating SHED suspension on the surface of the SHED cell membrane, wrapping the cell membrane in a mould after overnight cell adherence, culturing for 7d, extracting the mould, continuously culturing for 7d, folding and winding the SHED cell membrane to obtain dental pulp spline-shaped SHED cell polymer;

3) Washing the SHED cell polymer with PBS, decellularizing 3% Triton X-100 at 37deg.C for 72 h, and washing with PBS for 3min×3 times to obtain dental pulp bionic SHED-ECMS;

the use of said reed-ECMS for the preparation of a scaffold or material for promoting regeneration of pulp-dentin complex.

2. The method of claim 1, wherein said seed-ECMS has a porous and homogeneous extracellular matrix structure, provides a spatial scaffold for the growth of seed cells involved in dental pulp regeneration, including dental stem cells and jaw bone marrow stem cells, to promote migration and homing of the seed cells into the root canal of the tooth.

3. The method of claim 1, wherein the seed-ECMS is enriched in a molecular signal bioactive component of an early stage of pulp development, and is capable of inducing a process of restarting the damaged pulp tissue, activating the stem cells of dental origin, and promoting proliferation and differentiation of stem cells to form a pulp-dentin complex.

4. The method of claim 1, wherein the seed-ECMS is enriched in odontoblasts characteristic proteins, including DSPP and DMP1, capable of directionally inducing differentiation of seed cells into odontoblasts to form dental pulp-dentin complexes, reducing irregular calcification formation in root canals.

5. The preparation method according to claim 1, wherein the reed-ECMS is rich in a cytokine bioactive ingredient associated with pulp regeneration, and provides an optimal and most approximate physiological microenvironment for pulp regeneration to promote pulp-dentin complex regeneration.

6. A product for promoting regeneration of dental pulp-dentin complex comprising said seed-ECMS of claim 1 derived from extracellular matrix formed by secretion of seed.

7. The product of claim 6, wherein said shd is derived from human only physiologically SHED organ, deciduous dental pulp tissue, is easy to obtain, has unique and superior stem cell properties, including stronger in vitro expansion capacity, unique odontoblast differentiation, neuroblast differentiation and revascularization capacity, and more importantly, expresses a large number of pulp development related molecular signals, playing an important role in pulp development and regeneration.

8. The product of claim 6, wherein said she-ECMS is prepared from extracellular matrix secreted by odontogenic stem cells and is capable of dual biomimetic of pulp tissue morphology and bioactive components.

9. The product of claim 6, wherein said seed-ECMS morphological dimensions are personalized based on dental site and pulp cavity size, derived from purely natural cellular components.