CN116350842B - Application of biological hydroxyapatite combined with autologous periodontal ligament stem cells in immediate implantation of inflammation tooth extraction sites - Google Patents
Application of biological hydroxyapatite combined with autologous periodontal ligament stem cells in immediate implantation of inflammation tooth extraction sites Download PDFInfo
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- CN116350842B CN116350842B CN202310335662.3A CN202310335662A CN116350842B CN 116350842 B CN116350842 B CN 116350842B CN 202310335662 A CN202310335662 A CN 202310335662A CN 116350842 B CN116350842 B CN 116350842B
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- ligament stem
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Classifications
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Abstract
The invention discloses application of biological source hydroxyapatite combined with autologous periodontal ligament stem cells in immediate implantation of inflammation tooth extraction sites. Wherein, the fluoridated hydroxyapatite is taken as a framework, and periodontal ligament stem cells are attached to the surface of the fluoridated hydroxyapatite framework. The tissue engineering material obtained by combining the two can regulate PDLSCs growth microenvironment to be applied in an inflammatory environment, lighten the existing inflammation of the tooth extraction pit, promote PDLSCs osteogenic differentiation, widen the immediate planting technology to be applied to inflammatory environment indication, promote immediate planting success at an inflammatory tooth extraction site, and solve the aesthetic hot spot and difficulty of oral planting, namely reduction of labial alveolar bone absorption.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to application of biological hydroxyapatite combined with autologous periodontal ligament stem cells in immediate implantation of inflammatory tooth extraction sites.
Background
Immediate implantation is certainly an ideal repair for dentition defects (deletions) of tooth extraction caused by non-infectious factors such as trauma. Related researches prove that the immediate planting has a long-term success rate equivalent to that of the delayed planting, and has remarkable advantages in shortening the healing time of soft and hard tissues and reducing the times of patient visits. Immediate planting, however, also suffers from certain drawbacks, such as poorer predictability of labial alveolar ridge absorption (compared to delayed planting), thereby affecting restoration aesthetics. For this reason, labial alveolar ridge is mainly composed of a bundle bone nourished by periodontal ligament, and the bundle bone is absorbed after tooth extraction without periodontal ligament nourishment, thereby leading to labial alveolar ridge absorption. Based on this, the "root canal technique (root membrane technique, RM)", also called the root shield technique, is proposed in the related art, that is, a technique of intra-extraction implantation in the labial portion and periodontal ligament (periodontal ligament, PDL) of the reserved root in the same operation. Animal experiments show that in the instant implantation of the labial part of the reserved tooth root, osseointegration can be obtained between the implant and dentin of the labial tooth root, the buccal bone plate is preserved, the alveolar bone resorption is reduced, and the result is clinically verified. However, conventional RMs also have their inherent drawbacks, which, while aimed at preserving both root and periodontal membranes, limit the use and popularization of RMs at the risk of preserving root-related complications, such as root tip resorption, root exposure, etc.
In addition, the inflammatory periodontal ligament is not an indication for reserving the periodontal ligament in RM, and complications such as infection of reserved tooth roots on the cheek side, falling and shifting of reserved root slices, implant failure and the like can occur when the traditional RM technology is directly used in an inflammatory environment, so that final aesthetic failure is caused. However, the situation that the suffering teeth are required to be planted immediately but the patient is in an inflammatory state is often encountered clinically, so that the RM technology cannot be applied, which greatly limits the applicability of the traditional RM. Thus, how to widen RM technology remains a current major clinical difficulty in inflammatory environmental indications.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. Therefore, the invention aims to provide the application of the biological hydroxyapatite combined with autologous periodontal ligament stem cells in the immediate implantation of inflammatory tooth extraction sites. In the invention, the inventor successfully constructs the tissue engineering material which has anti-inflammatory property and can regulate and control inflammatory environment and is beneficial to the osteogenesis and differentiation of autologous periodontal ligament stem cells (seed cells) by combining biological hydroxyapatite and autologous periodontal ligament stem cells, thereby constructing an immune microenvironment which is beneficial to bone regeneration, widening the adaptability of the instant technology in the inflammatory environment, improving the success rate of instant planting in inflammatory tooth extraction sockets and solving the aesthetic hot spot and difficulty of oral planting which can not be overcome in the traditional technology, namely labial alveolar bone absorption.
In a first aspect of the present invention, there is provided a dental implant tissue engineering material comprising a fluorinated hydroxyapatite scaffold and periodontal ligament stem cells attached to the surface of the fluorinated hydroxyapatite scaffold.
In some embodiments of the invention, the fluorinated hydroxyapatite comprises particles of biogenic fluorinated hydroxyapatite prepared based on the preparation method in the prior chinese patented invention patent CN 102805879B. Of course, other biogenic fluorinated hydroxyapatite known in the art may be used by those skilled in the art, including but not limited to biogenic fluorinated hydroxyapatite prepared based on the preparation method of the prior chinese patented patent CN 102805879B.
Fluorine is an important trace element involved in bone reconstruction, and is often used for doping hydroxyapatite bone substitute materials for fluoride ion modification. Compared with unmodified hydroxyapatite, the fluorine modified hydroxyapatite has obviously improved acid resistance and strength. The biological source fluoridated hydroxyapatite particles adopted in the invention are obtained by adopting sodium fluoride solution hydrothermal treatment and a secondary high-temperature calcination technology (refer to the prior Chinese-patented invention patent CN 102805879B), and the tissue engineering material based on the biological source fluoridized hydroxyapatite particles also inherits the functions of promoting osteogenic differentiation of osteogenic related cells such as MG63, BMSCs and the like, promoting osseointegration and the like.
In some embodiments of the invention, the biological source includes, but is not limited to, bovine bone source, porcine bone source, coral bone source.
In some embodiments of the invention, the particle size of the fluorinated hydroxyapatite may be from 100 to 200 mesh.
In some embodiments of the invention, the fluorinated hydroxyapatite has a long columnar crystal morphology and has a pore structure.
In the prior art, it is not reported whether the pore-like structure of biogenic fluorinated hydroxyapatite would have an effect on PDLSC. The reason is that PDLSC, if present in the pore structure during cell division doubling, may undergo apoptosis, death, or pyro-death due to the pressure of the pore structure. Dead cells produce large amounts of inflammatory factors and immune cells and have negative effects on the normal functions of proliferation, differentiation, etc. of peripherally implanted PDLSC. In addition, because of the release of fluoride ion plasma after implantation of the biogenic fluorinated hydroxyapatite, it is currently unclear how the presence of fluoride ions affects PDLSC by reestablishing ion dissolution and precipitation equilibrium in the microenvironment. The invention combines the two for the first time, and finds that the two have no antagonistic relationship, and shows good combined effect after combined use, thereby greatly improving the meaning of combined use of the two.
In addition, the fluorine ion doping of the biogenic fluorinated hydroxyapatite in the invention causes the crystal structure of the hydroxyapatite on the biogenic hydroxyapatite to be changed from a sphere shape to a column shape, and can promote polarization of the crystal structure to M2 subtype by adjusting the adsorption proportion and the conformation change of fibronectin and fibrinogen in the crystal, and further activating macrophages through beta 1 integrin through intracellular PI3K/Akt signal paths, and secrete anti-inflammatory factors such as IL10, TGF beta and the like, thereby constructing an immune microenvironment which is favorable for bone regeneration, promoting PDLSCs osteogenic differentiation and promoting instant planting osseointegration and bone regeneration which keep periodontal membrane combined bone substitute materials.
In some embodiments of the invention, the content ratio of the fluorinated hydroxyapatite to the periodontal ligament stem cells in the tissue engineering material is 1 g:2-5×10 7.
In some embodiments of the invention, the tissue engineering material has a content ratio of fluorinated hydroxyapatite to periodontal ligament stem cells of 1g:2×10 7.
In some embodiments of the invention, the periodontal ligament stem cells are autologous-derived periodontal ligament stem cells.
In the present invention, the inventor found that retaining PDL (periodontal ligament) in immediate planting can make it stronger in osteogenesis, healthy PDL can promote osseointegration of implant, which is an important structure that should be retained in immediate planting, but periodontal ligament with inflammation cannot be applied in immediate planting. The tissue engineering material successfully overcomes the technical defect, so that periodontal membranes with inflammation can be brought into a range acceptable for immediate planting, and the application range of the immediate planting technology is greatly improved.
In a second aspect of the present invention, there is provided a method for preparing a tissue engineering material according to the first aspect of the present invention, comprising the steps of:
Taking periodontal tissues of a subject, digesting to obtain original periodontal ligament stem cells, subculturing, screening out healthy subcultured periodontal ligament stem cells, inoculating the healthy subcultured periodontal ligament stem cells to fluorinated hydroxyapatite, and incubating at 36-38 ℃.
In some embodiments of the invention, the fluorinated hydroxyapatite is substantially wetted by the basal medium.
In some embodiments of the invention, the incubation time is 1 to 2 minutes.
In some embodiments of the invention, the step further comprises performing a validated identification of passaged periodontal ligament stem cells.
The PDL promotes osseointegration and mainly derives from periodontal ligament stem cells (periodontal LIGAMENT STEM CELLS, PDLSCs), PDLSCs is a cell component which plays an important function in PDL, and is grown in a fibroblast form and a colony-like form in vitro, and has the multipotent of osteogenesis, cementogenesis, adipogenesis and angiopoiesis. For PDLSCs identification, however, the mesenchymal cell surface markers (i.e., CD105, CD90, CD73 positive, CD45, CD34, CD11b negative) are typically used to identify PDLSCs by flow cytometry.
In some embodiments of the invention, the primary periodontal ligament stem cells are socket-derived periodontal ligament stem cells.
In some embodiments of the invention, the culture environment of the periodontal ligament stem cells is: basal medium containing ascorbic acid, L-glutamine, penicillin, streptomycin and fetal bovine serum.
In some embodiments of the invention, the basal medium comprises an ALPHA-MEM medium.
In some embodiments of the invention, the culture environment of the periodontal ligament stem cells is: basal medium containing 100. Mu.M/L ascorbic acid, 0.292mg/mL L-glutamine, 100U/mL penicillin, 100U/mL streptomycin and 10% fetal bovine serum.
PDLSCs characteristics are affected by a variety of factors, such as ① host cell source: PDLSCs from different sources have different biological properties. The primary tooth source PDLSCs was stronger in proliferation capacity, adipogenic differentiation capacity and osteogenic differentiation capacity than the permanent tooth source. Whereas the extracted socket source has a stronger proliferation capacity and osteogenic differentiation capacity than PDLSCs, which is a source of root surface. ② Age of host: estrogens maintain PDLSCs dryness, and the older the host is, the worse the PDLSCs dryness and the multipotent differentiation. ③ Growth microenvironment: the growth microenvironment has very remarkable influence on the characteristics of PDLSCs, such as the characteristics of PDLSCs caused by different microelements contained in a culture medium, and the oxygen concentration, the inoculated cell density and the like of the culture environment are also influenced. In vivo environments, the local growth microenvironment also includes various cytokines and biological materials. In the invention, the biological source fluoridated hydroxyapatite particles are combined, so that the expression of the osteogenic differentiation related genes Runx2 and Osterix in PDLSCs can be induced to be increased, and the osteogenic differentiation of PDLSCs is promoted. In clinical practice, the source and age of the host cell are often unalterable factors. Therefore, it is very important to regulate the growth microenvironment to promote the differentiation of host tooth socket autologous PDLSCs in the osteogenic direction to promote osseointegration.
In a third aspect, the invention provides the use of the tissue engineering material according to the first aspect of the invention for the preparation of an immediate dental implant product.
In some embodiments of the invention, the suitable population of immediate dental implant products includes a population in need of immediate dental implant at an inflammatory extraction site.
The physiological process of inflammation is often accompanied by the release of inflammatory cell pro-inflammatory factors and the subsequent infiltration of immune cells, and the storm effect of inflammatory factors is accompanied by the release of cytokines such as matrix metalloproteinase, which may cause promotion of bone fracture, and cause bone resorption to inhibit bone formation, so that the regeneration balance is damaged, and the method is unfavorable for the fixation of osteoblasts or seed cells. That is, inflammation itself causes not only inflammation granulation but also disruption of physiological balance of the surrounding microenvironment. The inflammation granuloma tissue is thoroughly removed from the inflammation tooth extraction pit, only the existing inflammation granuloma tissue is removed, but the inflammatory cells, particularly inflammatory factors, in the microenvironment around the inflammation cannot be completely removed, and the destruction of the inflammation on the local microenvironment balance cannot be immediately reversed through a plurality of researches.
In addition, after removal of the inflamed granulation tissue, the body needs to act as an immune system to reverse the inflammatory microenvironment to facilitate regeneration and reconstruction, which requires healing time. The instant planting is widely favored because of the time specificity, the instant tooth extraction can provide a planting technology for patients, and the waiting time of the patients is reduced. However, the reversal of inflammatory microenvironment in inflammatory sockets requires healing time, which has been a contraindication for immediate implantation.
In the invention, by the combined use of the biogenic fluoridated hydroxyapatite and the autologous PDLSCs, not only the effective existence of the periodontal ligament stem cells is ensured, but also the inflammatory microenvironment is reversed, and the bone regeneration balance is rebuilt. This is the technical focus and difficulty of using RM in inflammatory environments to regulate PDLSCs growth microenvironment that cannot be overcome in the prior art.
In some embodiments of the invention, the method of using the dental instant implant product is: the dental immediate implant product is filled between the labial bone wall of the socket and the labial implant.
In some embodiments of the invention, a jumping gap is left between the labial bone wall of the socket and the labial side of the implant, the tooth implant product immediately filling in the jumping gap.
In some embodiments of the invention, the width of the jump gap is 2mm or more.
In some embodiments of the invention, after filling is completed, a healing abutment or temporary crown of the closed socket is worn and sutured.
In some embodiments of the invention, the filling means includes, but is not limited to, loading the filling using a syringe. Of course, those skilled in the art can construct various forms of implantation, including changing the form thereof into an injectable tissue engineering material, based on the actual use requirements, to achieve similar technical effects.
In a fourth aspect, the present invention provides the use of a tissue engineering material according to the first aspect of the present invention for the preparation of a bone repair product.
In some embodiments of the invention, the method of using the bone repair product is: the bone repair product is coated or filled on the damaged or to-be-repaired part of the bone.
The beneficial effects of the invention are as follows:
The invention constructs a tissue engineering material containing autologous healthy PDLSCs and biogenic fluorinated hydroxyapatite, which can be used for immediate implantation of inflammatory tooth extraction sites, can regulate and control PDLSCs growth microenvironment to be applied in inflammatory environment, lighten existing inflammation of tooth extraction sockets, promote PDLSCs osteogenic differentiation, widen the immediate implantation technology to be applied to inflammatory environment indication, promote immediate implantation success of inflammatory tooth extraction sites, and solve aesthetic hot spots and difficulties of oral implantation, such as reduction of labial alveolar bone absorption.
Drawings
FIG. 1 shows PDLSCs monoclonal formation of human periodontal ligament stem cells (PDLSCs) by primary isolation and culture for 12 days; scale bar is 100 μm and B is a partial enlargement of A.
FIG. 2 is a diagram of PDLSCs cell immunostaining, wherein A is a graphic representation of the positive expression of vimentin, and B is a graphic representation of the non-expression of CK 18; the scale bar is 100 μm.
FIG. 3 is a alizarin red staining chart of PDLSCs; the scale bar is 100 μm.
FIG. 4 is a chart of PDLSCs oil red O staining; the scale bar is 100 μm.
FIG. 5 shows the results of flow cytometry detection of primary extracted cultured cell surface markers derived from human periodontal ligament.
FIG. 6 is a scanning electron microscope contrast diagram of BHAp crystals and FBHAp crystals.
Fig. 7 is an EDX energy spectrum of FBHAp and BHAp.
Figure 8 is an XRD diffractogram of FBHAp and BHAp.
FIG. 9 is a FTIR absorption spectrum of FBHAp and BHAp.
FIG. 10 shows the results of alizarin red staining of PDLSCs after 21d osteoinductive culture.
FIG. 11 shows semi-quantitative alizarin red staining of PDLSCs after osteoinductive culture for 21 d.
FIG. 12 shows ALP activity PDLSCs after osteoinductive culture 21 d.
FIG. 13 shows the results of alizarin red staining of PDLSCs cultured with direct contact FBHAp of macrophage supernatant as culture medium, where-indicates no osteogenesis and +indicates osteogenesis.
FIG. 14 shows ALP activity (A) and alizarin red staining semi-quantitative results (B) of PDLSCs cultured with directly contacted FBHAp macrophage supernatant as a culture medium.
FIG. 15 shows the expression of PDLSCs bone-related genes cultured with directly contacted FBHAp macrophage supernatant as a culture medium.
FIG. 16 shows the effect of the tissue engineering material of the present invention on promoting critical bone defects of rat skull, wherein A is a microCT image, B is bone volume fraction (BV/TV), and C is new bone formation rate (NB%).
FIG. 17 is a chart of a histopathological TRAP stain for FBHAp repair of rabbit skull defects.
FIG. 18 is a comparison of TRAP activity of RAW264.7 monocytes seeded at FBHAp and BHAp surfaces.
FIG. 19 is a comparison of IL6 and TNFα mRNA expression levels following FBHAp and BHAp surface-seeded RAW264.7 monocytes.
FIG. 20 is a quantitative analysis of TRAP activity of RAW264.7 monocytes seeded on FBHAp and BHAp surfaces.
FIG. 21 shows the expression ratio of cell surface marker F4/80 characteristic of M2 type RAW264.7 monocytes inoculated at FBHAp and BHAp surface, wherein red line is BHAp surface-inoculated RAW264.7 monocytes and blue line is FBHAp surface-inoculated RAW264.7 monocytes.
FIG. 22 is a scanning electron microscope SEM morphology of FBHAp and BHAp surface seeded macrophages.
FIG. 23 shows the expression of macrophage-type surface markers M0, M1 and M2 after human peripheral blood-derived macrophages are inoculated into different experimental group materials and cultured for 72 hours.
Fig. 24 is a pre-operative case of an application of tissue engineering material in the immediate implantation of an inflammatory extraction site.
Fig. 25 shows the application of tissue engineering material in the immediate implantation of inflammatory extraction sites during and after surgery.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples were either commercially available from conventional sources or may be obtained by prior art methods unless specifically indicated. Unless otherwise indicated, assays or testing methods are routine in the art.
Tissue engineering material capable of being used for immediate implantation of inflammation tooth extraction sites
The present embodiment provides a tissue engineering material capable of being used for immediate implantation of an inflammatory extraction site, the tissue engineering material comprising: the bone made of the fluorinated hydroxyapatite and the autologous healthy periodontal ligament stem cells adhered to the fluorinated hydroxyapatite for growth.
The preparation method of the tissue engineering material specifically comprises the following steps:
(1) Obtaining and culturing autologous healthy periodontal ligament stem cells (hPDLSC):
patients who need immediate implantation and have inflammatory tooth extraction sites are subjected to extraction of wisdom teeth before immediate implantation, and periodontal ligament stem cells are isolated and cultured.
The specific separation and culture method comprises the following steps: after removal of wisdom teeth, 1/3 of periodontal tissue in the removed wisdom tooth roots was scraped, minced, and digested at constant temperature for 1 hour at 37℃using a mixture of 3mg/mL (final concentration) of type I collagenase and 4mg/mL (final concentration) of dispase. Digestion was terminated, and the cells were transferred to a basal medium (ALPHA-MEM medium) containing ascorbic acid at a final concentration of 100. Mu.g/mL, L-glutamine at 0.292mg/mL, penicillin at 100U/mL, streptomycin at 100U/mL, and 10% fetal bovine serum, cultured, and the colony cells were collected and transferred to 3 to 5 passages for subsequent use. After passage, periodontal ligament stem cells with good growth state are digested to obtain cell suspension, and the cell suspension is inoculated into a 6-hole plate at the density of 5X 10 5 cells/mL after counting. Detection of surface markers was performed using a flow cytometer. Periodontal ligament stem cell surface markers include: CD105, CD166, CD44, CD90, HLA-DR, CD34, CD45. Among them, CD105, CD166, CD44, CD90 showed positive and HLA-DR, CD34, CD45 showed negative, indicating successful acquisition of autologous healthy periodontal ligament stem cells.
Among them, the flow cytometry detection method is performed with reference to the routine procedures in the art, and antibodies for detection of CD105, CD166, CD44, CD90, HLA-DR, CD34, CD45 are purchased from Biolegend company (USA).
Further, the obtained autologous healthy periodontal ligament stem cells were subjected to identification experiments such as clone formation, tissue origin identification (cell immunostaining), and multi-directional differentiation to confirm the correctness of the obtained cells.
The results are shown in FIGS. 1 to 5.
FIG. 1 shows PDLSCs monoclonal cells formed after 12 days of culture. Cell immunostaining demonstrated that PDLSCs monoclonal vi mentin formed was positive for expression and did not express CK18 (red positive, blue dapi-stained nuclei), indicating that the extracted cells were of mesenchymal (mesenchymal) origin rather than epithelial origin. Furthermore, the resulting PDLSCs monoclonal had a multipotent differentiation potential, in which alizarin red and oil red O staining demonstrated that the resulting PDLSCs monoclonal was able to differentiate into osteoblasts or adipocytes upon culture under osteogenic induction (osteogenic induction fluid containing osteogenic induction components (10 -7 M dexamethasone, 10mM sodium beta-glycerophosphate, 50 μm ascorbic acid)) and adipogenic induction conditions (adipogenic induction fluid containing adipogenic induction components 10 -6 M dexamethasone, 10ng/mL insulin, 0.5 μm IBMX, 0.2mM indomethacin), indicating that the cells had a multipotent differentiation potential characteristic of stem cells. In addition, the results of flow cytometry showed that CD34 (0.68%), CD45 (0.94%), CD105 (99.24%), CD 166 (99.51%), CD44 (98.94%), CD90 (99.28%), and cell surface marker characteristics conforming to PDLSCs, thereby confirming the extracted cell composite periodontal ligament stem cell surface marker characteristics, indicating that autologous healthy periodontal ligament stem cells were obtained successfully by the above-described isolation culture.
(2) Cell seeding on a fluoridated hydroxyapatite scaffold:
① Preparation of a fluoridated hydroxyapatite skeleton:
The biological source fluoridated hydroxyapatite particles (FBHAp with the particle size of 100-200 meshes) are prepared according to the preparation method in the prior Chinese patent CN 102805879B.
The resultant biogenic fluorinated hydroxyapatite particles (FBHAp) and BHAp (FBHAp without fluorine) were observed under electron microscopy and physically and chemically characterized using EDX, XRD and FTIR, respectively.
The results are shown in FIGS. 6 to 9.
It was found that under electron microscopy, FBHAp crystals were transformed from pre-preparation spheres to post-preparation long columns, and that the tertiary pore structure of biological bone-derived FBHAp was observed to be preserved, and the pore size in FBHAp was small (relative BHAp). Whereas EDX, XRD and FTIR characterization patterns confirm that fluoride ion substituted hydroxyl (OH) was successfully incorporated into HAp (hydroxyapatite), FBHAp was obtained. Among them, the EDX spectroscopy analysis of FBHAp showed that the atomic percentages of the elements BHAp and FBHAp, and Ca, P, O, C was found to be the main constituent element of BHAp and FBHA, and trace amounts of Na and Mg were also detected to different extents in the BHAp and FBHAp materials. The carbon content of FBHAp is increased compared to BHAp. Comparison of XRD diffraction spectra of BHAp and FBHAp with the stoichiometric hydroxyapatite standard diffraction spectrum card (JCPCDS card # 09-0432) shows that the diffraction peaks of sample crystal faces (002), (102), (211), (112), (300), (202), (310), (222), (213) are consistent with the standard card, and no characteristic peaks of other phases are detected. After doping with fluoride ions, FBHAp XRD diffraction peaks drift toward a high diffraction angle as a whole, and the trend is particularly obvious in diffraction peaks corresponding to (300) crystal faces and (211) crystal faces. FBHAp, the characteristic phosphate absorption peaks P-Ov 1 (962 cm -1)、ν2(473cm-1), v 3 (1092 and 1046cm -1) and v 4 (602 and 218 cm -1) are substantially coincident with FBHAp at BHAp, the carbonate absorption peaks C-O v 2 (874 cm -1) and v 3 (1461-1410 cm -1) are obviously stronger than BHAp in FBHAp, and the double absorption peak prompt material in the wave band belongs to B-type carbonate hydroxyapatite (Carbonated hydroxyapatite, CHA), namely carbonate in the carbonate part is replaced by carbonate in the hydroxyapatite. In addition, after doping with fluoride ions, the hydroxyl absorption peaks at 3572cm -1 and 634cm -1 in FBHAp absorption spectrum are obviously weakened or even disappeared, and instead, absorption peaks at 3538cm -1 and 745cm -1 indicate the formation of OH-F and OH-F-HO valence bonds.
② Planting autologous healthy periodontal ligament stem cells:
In a sterile operation table, the biogenic fluorinated hydroxyapatite particles are placed in a 6-well plate, the biogenic fluorinated hydroxyapatite particles are fully wetted with the culture medium according to the amount of using 2mL of basic culture medium for each 0.25g of the biogenic fluorinated hydroxyapatite particles, and the autologous healthy periodontal ligament stem cells are inoculated on the biogenic fluorinated hydroxyapatite particles according to the amount of 5X 10 6 autologous healthy periodontal ligament stem cells/0.25 g of the biogenic fluorinated hydroxyapatite particles, and after incubation at a constant temperature of 37 ℃, the tissue engineering material capable of being used for immediate implantation of inflammatory extraction sites is obtained.
In vitro effect verification of tissue engineering materials
The tissue engineering material in the above example was cultured with an osteogenic induction solution containing osteogenic induction components (10 -7 M dexamethasone, 10mM sodium beta-glycerophosphate, 50. Mu.M ascorbic acid) at a constant temperature of 37℃until day 21, and was subjected to alkaline phosphatase (ALP) activity assay (alkaline phosphatase assay kit, commercially available from Nanjing), alizarin red staining (Sigma, USA) and alizarin red semi-quantitative analysis. A blank (PDLSCs not co-cultured with FBHAp) was set.
The results are shown in FIGS. 10 to 12.
It can be found that PDLSCS ALP activity level of FBHAp co-culture in the tissue engineering material is higher, and the staining and quantitative result of alizarin red shows that the osteogenesis and mineralization capability is stronger, and FBHAp can promote PDLSCs differentiation to the osteogenesis direction in vitro. Thus, it was revealed that PDLSCs had a strong osteogenic differentiation after co-culture with FBHAp, and FBHAp was able to promote PDLSCs differentiation in the osteogenic direction in vitro.
The inventors have also found that macrophages can further promote their attachment PDLSCs to bone after direct contact FBHAp. In this regard, the inventors performed the following experimental verification, specifically: macrophages were inoculated into plates containing 0.2: 0.2g FBHAp at a density of 10 6 cells/well, respectively, and incubated for 72h, and the supernatants were collected by centrifugation. The supernatant (directly contacted with FBHAp macrophage supernatant) is used for preparing a culture solution, and the specific preparation method is as follows: taking the supernatant according to the following ratio of 1:1 and an osteogenesis inducing culture medium are mixed and used for inducing PDLSCs osteogenesis differentiation, after 14d induction, the gravel-shaped mineralized nodules formed by shooting are used for carrying out semiquantitative analysis of alizarin red staining and quantitative analysis of ALP activity. As a control, a medium without the above osteoinductive liquid was used.
The results are shown in fig. 13 and 14. Human periodontal ligament stem cells co-cultured with FBHAp aggregate and grow and form a large number of mineralized nodules, as compared to the group without FBHAp, as compared to the tissue engineering material without the present invention. Further based on the semi-quantitative test results, it was shown that the alkaline phosphatase activity and mineralization level of human periodontal ligament stem cells co-cultured at FBHAp containing an osteoinductive component (reduced) were higher than those of cells in the osteoinductive component medium as a control, suggesting that the osteogenic differentiation and mineralization of human periodontal ligament stem cells were stronger than those of the control in the co-culture environment with the material. In contrast, whether co-culture with the material had no significant effect on cellular alkaline phosphatase levels in basal medium without osteogenic inducing component (uninduced) (no significant statistical difference from control).
The expression levels of the cultured PDLSCs osteogenesis-related genes RUNX2, ALP, BSP and ATF4 mRNA (without induction (0 d), induction 4d and 7 d) were further examined using PCR amplification.
The results are shown in FIG. 15. It can be seen that q-RT-PCR detection shows that PDLSCs (dark panel) osteogenic related genes RUNX2, ALP, BSP and ATF4mRNA expression levels were co-cultured for 7d in osteogenic induction environment directly contacted with FBHAp macrophage supernatant compared to control (light panel) (p <0.05, difference statistically significant). Thus, it was demonstrated that PDLSCs cultured from macrophage supernatant in direct contact with FBHAp had a strong osteogenic differentiation and increased mRNA expression levels of the osteogenic related genes RUNX2, ALP, BSP and ATF 4.
Animal test (in vivo) effect verification of tissue engineering materials
(1) Promotion of early osteogenesis of critical bone defects in rat skull by tissue engineering materials:
and repairing the critical bone defect of the skull of the experimental animal by using the material, and taking a microCT after finishing the repair for three months. A blank control was set and controls were performed using FBHAp alone.
The results are shown in FIG. 16.
The results show that the tissue engineering material (i.e. FBHAp + PDLSCs) in the above embodiment has a remarkable promoting effect on early osteogenesis of critical bone defects of the rat skull.
(2) Repairing effect and anti-inflammatory effect of tissue engineering materials on rabbit skull defects:
the critical bone defect of the skull of the New Zealand white rabbit of the experimental animal is repaired by using the materials, and a sample is taken after three months to remove ore tissue sections and fix the TRAP for dyeing.
The results are shown in FIG. 17.
From the FBHAp histopathological TRAP staining graph for repairing rabbit skull defects, it was seen that FBHAp material recruited intravascular TRAP-positive macrophages to the surrounding material and activated TRAP activity by direct contact.
In addition, to further verify the anti-inflammatory effect of tissue engineering materials, the inventors inoculated RAW264.7 monocytes on FBHAp surfaces, stained with TRAP, and detected the expression level of inflammatory factors (IL 6 and tnfα) mRNA using PCR. At the same time, flow cytometry was used to further examine the expression of cell surface marker F4/80 characteristic of the M2 type of inoculated RAW264.7 monocytes. BHAp was used as a control.
The results are shown in FIGS. 18 to 21. The results show that FBHAp surface inoculated RAW264.7 monocytes have lower TRAP activity, reduced IL6 and TNF-alpha gene expression, and increased expression ratio of cell surface marker F4/80 characteristic of M2 type. Therefore, the tissue engineering material can be used for regulating the inflammatory microenvironment to adjust the immunity direction favorable for bone formation.
In addition, scanning electron microscope morphological analysis is further carried out on FBHAp surface macrophages, and flow cytometry is used for detecting macrophage parting surface markers, wherein M1 is PC7-HLA-DR; m2 is M2a PE-CD 206 and M2c APC-CD 163. Wherein, when the morphology and micro morphology of the macrophage population are analyzed based on morphology, the cell aspect ratio is defined as polarized cells when it is > 1.5.
The results are shown in FIGS. 22 to 23.
From the electron microscope, it can be found that macrophages are mainly polarized on FBHAp surfaces, and columnar crystal structures of FBHAp after fluorine doping promote macrophage expansion and pseudopodia formation on the surfaces of materials, so that the proportion of polarized cells (aspect ratio is more than 1.5) is increased. Furthermore, after 72h incubation of human peripheral blood-derived macrophages on FBHAp, after detection of macrophage-parting surface markers based on flow cytometry, FBHAp stimulated elevation of both macrophage M2 surface marker CD206 and CD163 expression was found.
In conclusion, the repairing effect and anti-inflammatory effect of the tissue engineering material on rabbit skull defects can be demonstrated. Application effect of tissue engineering material in immediate implantation of inflammation tooth extraction site
In the test example, the actual application effect of the tissue engineering material in the embodiment in the immediate implantation of the inflammation tooth extraction site is tested and verified, and the specific test method is as follows:
According to the conventional immediate implantation surgery, local anesthesia disinfection towel is carried out on patients needing immediate implantation and having inflammatory tooth extraction sites. The inflammatory granulation tissue in the inflammatory dental socket is thoroughly removed, and an implant is implanted in the correct three-dimensional position. After implantation, a jump gap of more than 2mm is reserved between the labial bone wall of the tooth extraction socket and the labial of the implant. Implantation is performed using the tissue engineering material in the above embodiments to fill the jump gap. After filling, the closed dental extraction pit is worn on a healing abutment or a temporary crown and is sutured.
Specific cases are shown in fig. 24 to 25.
In case of 45-year-old male, the upper left middle incisors are simultaneously planted in a parallel way when being removed due to severe periodontitis, and the lower left wisdom teeth are removed before operation to extract and culture periodontal ligament stem cells. The left upper middle incisor of the patient is removed by minimally invasive, the tooth extraction pit is scratched, the implant is implanted immediately, and the collagen membrane is covered by the implant tissue engineering material. The implant and tissue engineering materials complete osseointegration in half a year after operation, and the final repair effect is good.
Taken together, the results show that the tissue engineering material in the above embodiment can be used for clinical application of immediate implantation of inflammatory tooth extraction sites, and has remarkable and effective application effects.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (3)
1. An application of tissue engineering materials in preparing instant tooth planting products, wherein applicable people for the instant tooth planting products comprise people needing instant planting at an inflammation tooth extraction site;
The tissue engineering material consists of a fluorinated hydroxyapatite skeleton and periodontal ligament stem cells attached to the surface of the fluorinated hydroxyapatite skeleton;
the periodontal ligament stem cells are autologous periodontal ligament stem cells;
in the tissue engineering material, the content ratio of the fluoridated hydroxyapatite to the periodontal ligament stem cells is 1 g:2-5 multiplied by 10 7;
the application method of the tooth immediate planting product comprises the following steps: the dental immediate implant product is filled between the labial bone wall of the socket and the labial implant.
2. The use according to claim 1, wherein the tissue engineering material is prepared by the following method:
taking periodontal tissues of a subject, digesting to obtain original periodontal ligament stem cells, subculturing, screening out healthy subcultured periodontal ligament stem cells, inoculating the healthy subcultured periodontal ligament stem cells to fluoridated hydroxyapatite moistened by a basic culture medium, and incubating at 36-38 ℃.
3. Use according to claim 2, characterized in that the incubation time is 1-2 min.
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| CN101579537A (en) * | 2009-07-07 | 2009-11-18 | 中国人民解放军第四军医大学 | Surface treatment method of tissue-engineered bioactive implant |
| CN102805879A (en) * | 2012-07-20 | 2012-12-05 | 陈卓凡 | Preparation method for biogenic fluorinated porcine hydroxyapatite (FPHA) bone substitution material |
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| CN102755667B (en) * | 2012-07-05 | 2014-06-25 | 中国人民解放军第四军医大学 | Preparation method and application of tissue-engineering human tooth root implantation material |
| CN105536061A (en) * | 2015-09-30 | 2016-05-04 | 王松灵 | Biological tooth root scaffold/cell/diaphragm complex containing bionic periodontal membrane, and tissue engineering construction method thereof |
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| CN108753702A (en) * | 2018-06-11 | 2018-11-06 | 南京泰盛生物科技有限公司 | A kind of Periodontal ligament stem cell subgroup and its dryness detection method by cell surface marker specific enrichment |
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| CN102805879A (en) * | 2012-07-20 | 2012-12-05 | 陈卓凡 | Preparation method for biogenic fluorinated porcine hydroxyapatite (FPHA) bone substitution material |
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