WO2023135481A1 - Fabrication and evaluation of dentin powder surface modified with alginate on proliferation of dental pulp stem cells (in vitro) and regeneration of dentin organ and immunogenicity (in vivo) - Google Patents

Fabrication and evaluation of dentin powder surface modified with alginate on proliferation of dental pulp stem cells (in vitro) and regeneration of dentin organ and immunogenicity (in vivo) Download PDF

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WO2023135481A1
WO2023135481A1 PCT/IB2022/062774 IB2022062774W WO2023135481A1 WO 2023135481 A1 WO2023135481 A1 WO 2023135481A1 IB 2022062774 W IB2022062774 W IB 2022062774W WO 2023135481 A1 WO2023135481 A1 WO 2023135481A1
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dentin
alginate
tissue
immunogenicity
groups
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PCT/IB2022/062774
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French (fr)
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Melika MANZARPOUR
Hengameh Bakhtiar
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Manzarpour Melika
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/734—Alginic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3865—Dental/periodontal tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

Definitions

  • This invention is about the fabrication and evaluation of dentin powder surface modified with alginate on the proliferation of dental pulp stem cells (in vitro) and also the regeneration of dentin organs and immunogenicity (in vivo) root canal treatment of necrosis, traumatized or extended decayed. so we make material to promote dentin-pulp-like tissue to complete the maturation of the apex and thickening wall of the root which degraded during this procedure.
  • surface modification of demineralized dentin powders with alginate is beneficial to improve the mechanical properties of the surface and shows better biocompatibility and minimum immunogenicity which can be used for dentin-pulp-like tissue.
  • the embodiments herein provide a dental and medical biomaterial and its use for sealing and/or filling the tooth and bone cavities.
  • the calcium salt, calcium oxide, calcium silicate, and calcium phosphate compounds are mixed with a water-based solution, and a bioactive phosphate and calcium-enriched mixture are prepared.
  • the blend comprises a high concentration of water-soluble calcium and phosphate and consequently forms hydroxyapatite during and after setting.
  • the dental/medical biomaterial is biocompatible, antibacterial, and capable of creating an effective seal against the reentrance of microorganisms into the filled cavity.
  • the biomaterial is compatible to handle and set in an aqueous environment and stimulating soft/complex tissue healing/generation/regeneration.
  • Biomaterial systems e.g., gel scaffolds, are used in vivo to recruit immune cells and promote their activation towards a non-inflammatory phenotype, thereby leading suppression of inflammation.
  • the compositions and methods are useful to reduce the severity of autoimmunity, chronic inflammation, allergy, and periodontal disease.
  • the invention provides methods, apparatus, and kits for regenerating dental tissue in vivo that are useful for treating a variety of dental conditions, exemplified by the treatment of caries.
  • the invention uses tissue scaffold wafers, preferably made of PGA, PLLA, PDLLA or PLGA dimensioned to fit into a hole of the corresponding size drilled into the tooth of the subject to expose dental pulp in vivo.
  • the tissue scaffold wafer further comprises calcium phosphate and fluoride.
  • the tissue scaffold wafer may be secured into the hole with a hydrogel, a block of cement, or other suitable material.
  • Either the wafer or the hydrogel or both contain a morphogenic agent, such as a member encoded by the TGF- ⁇ supergene family, that promotes regeneration and differentiation of healthy dental tissue in vivo, which in turn leads to remineralization of dentin and enamel.
  • the tissue scaffold may further include an antibiotic or anti-inflammatory agent.
  • the dental pulp is susceptible to various injuries such as bacteria invasion, caries, and trauma. These factors lead to pulp necrosis, disruption of dentin formation, and prohibition of root development by interfering with the normal physiology of the pulp.
  • the current common clinical treatment attempts to remove pulp necrosis, prevent the inflammation of the apical foramen and partially restore the normal function of the tooth.
  • this treatment due to limitations such as difficulty in establishing apical seals in open apex tooth and halting the dentinal wall's development insults to render the walls thin and increase the possibility of tooth fracture.
  • This invention is obtaining a biocompatible and biodegradable biomaterial from bovine dentin under physical and chemical modifications, that can induce the process of tissue regeneration.
  • the dental pulp is susceptible to various injuries such as bacteria invasion, caries, and trauma. These factors lead to pulp necrosis, disruption of dentin formation, and prohibition of root development by interfering with the normal physiology of the pulp.
  • the current common clinical treatment attempts to remove pulp necrosis, prevent the inflammation of apical foraman and partially restore the normal function of the tooth.
  • this treatment due to limitations such as difficulty in establishing apical seals in open apex tooth and halting the dentinal walls development, insults to render the walls thin and increase the possibility of tooth fracture.
  • Dentin is a connective tissue, rich in collagen type 1 and bioactive proteins which are release from dentinal tubules under certain conditions, including controlled demineralization.
  • growth factors have been found to play a role in tissue repair, cell distinction, and anti-inflammatory processes and it has been proven that these growth factors can be extracted from the surface-modified dentin.
  • This invention is obtaining a biocompatible and biodegradable biomaterial from bovine dentin under physical and chemical modifications, that can induce the process of tissue regeneration. This can improve the root treatment process through pulp and dentin complex regeneration in the treatment of vital permanent immature open apex teeth .
  • surface-modified dentin powder with alginate due to the characteristics of this natural polymer such as biocompatibility, biodegradability, and extraordinary plasticity as a suitable scaffold improves dentin-pulp complex regeneration.
  • Dentin is an integrated tissue, rich in collagen type 1 and bioactive proteins which are release from dentinal tubules under certain conditions, including controlled demineralization. Among these proteins, several growth factors have been found to play a role in tissue repair, cell distinction, and anti-inflammatory processes and it has been proven that these growth factors can be extracted from surface-modified dentin.
  • Surface-modified dentinal powder with alginate due to the characteristics of this natural polymer such as biocompatibility, biodegradability, and extraordinary plasticity as a suitable scaffold improves dentin-pulp complex regeneration.
  • This study is experimental and its purpose is to investigate the effect of surface-modified bovine dentin powder with alginate in groups of 150-250, 250-500, and 500-1000 micrometers on the proliferation of dental pulp stem cells in laboratory conditions (in vitro) and regeneration of dentin-pulp like structure and immunogenicity in animal (in vivo).
  • FTIR-ATR test different wavelengths (infrared) are irradiated to the samples and the amount of absorption of each wavelength is measured, and in this way, the curve of the amount of light absorption according to different wavelengths is obtained for each sample. Considering that amino acids and especially telopeptides have maximum absorption at a specific wavelength, in this way, the number of remaining telopeptides was compared. With this test, we reached the quantitative measurement of the samples.
  • This test shows the activity of mitochondria in the cell and has a direct relationship with cell growth and cell survival, which was done according to the protocol of MTT dye.
  • a solution of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl 2H-MTT tetrazolium bromide (Merck, Germany) was prepared in PBS with a concentration of 5 mg/ml.
  • DPSC cells are planted in 96-well plates (6000 cells per well) in the vicinity of xenograft bovine dentin powder.
  • the culture medium containing the tested compounds is changed and replaced with a fresh culture medium containing MTT with a concentration of 0.5 mg/ml, then the cells are placed in an incubator with a temperature of 37 degrees Celsius and 5% CO2 for 3 hours.
  • the insoluble crystals of formazan produced by living cells are dissolved using dimethyl sulfoxide and the quantity of formazan obtained is determined by the spectrophotometric method by a Microplate reader at a wavelength of 570 nm.
  • Rats were first anesthetized using ketamine xylazine then, in fully sterile conditions, using the scalpel, the long cut was given in rat Calvaria, and using trephine Bur, a defect with a 7mm diameter was created and then the resulting defect was filled with prepared powders and the area was closed with suture and the mice were returned to the maintenance site. After 8 weeks, the rats were sacrificed via anesthesia medicine overdose, and the area under investigation was separated from the skull and placed immediately inside the formalin solution for 2 days.
  • the samples were placed in EDTA for decalcification for 4 weeks and a block was prepared from them using Para film and histological slides were obtained.
  • the obtained slides were stained with H&E stain for histological examination.
  • To examine the histology of the obtained slides two pathologists examined the slides with an optical microscope. The amount of newly formed bone tissue compared to the total defect created was investigated. To distinguish between newly formed bone with dentin and bone without cells, the indicator of osteocytes inside the lacuna was used.
  • the tissue response adjacent to the implanted samples was carefully examined and ranked in terms of the presence of inflammatory cells, fibrosis, bleeding, necrosis, and the creation of new blood vessels.
  • the use of surface-modified xenograft dentin powder with alginate to improve the structural features of dentin can be a key to solving the limitations of regenerative endodontics.
  • surface-modified dentin powder with alginate was investigated on the proliferation of dental pulp stem cells and restoration of dentinal structure and immunogenicity.
  • the dentin powder size index separated the studied groups into three groups: 1. 150-250 micrometers, 2. 250-500 micrometers, and 3. 500-1000 micrometers.
  • the studied groups that were demineralized by 37% phosphoric acid for 30 seconds, are dentins containing numerous non-collagen proteins that include growth factors such as BMP, as soon as the dentin is demineralized, the dentin tubules become more dilated and can act as a route to flow these basic proteins.
  • BMP growth factors
  • FE-SEM and EDX were tested with FE-SEM and EDX. Based on the qualitative results obtained, the complete removal of smear layer and smear plug in the opening of dentin tubules and the exposure of collagen fibers and the opening of tubules indicated the success of demineralization in three groups.
  • the composition of dentin in terms of volume in the mineral part is 70% by weight and the organic part is 20% and the rest is water. Based on the quantitative results of EDX analysis, the decrease in the weight percentage of calcium and phosphate mineral elements, which are the constituent elements of the mineral structure of dentin, and the increase in the weight percentage of carbon in the organic part, which is one of the constituent elements of the organic structure of dentin, indicated the success of the demineralization process in every Three groups were studied.
  • Hydroxyapatite crystals which are one of the basic structures of dentin, can cover the telopeptides of dentinal collagens to prevent their removal in the procedure of pepsin digestion. For this reason, after successful demineralization, the dentin powders were subjected to the Atelopeptide procedure. Then, the present samples were surface modified with alginate according to the mentioned protocol. Specific peaks of the mannuronic acid functional group in wave 884 and uronic acid functional group in wave 939 are specific wavelengths of alginate in the FTIR study. According to the data obtained from all three studied groups and dentin powder compared to alginate, the creation of a peak in the mentioned wavelengths was observed in the groups of surface-modified with alginate.
  • the general concept of tissue engineering is to combine a matrix or a Scaffold with viable cells to achieve a cell-biomaterial structure to stimulate tissue repair and regeneration by using this structure.
  • DPSCs dental pulp stem cells
  • MTT staining has shown that on the third day, the proliferation of stem cells placed on the dentinal matrix between the 150-250 ⁇ m group compared to the 250-500 ⁇ m group and also the 250-500 ⁇ m group compared to the 500-1000 ⁇ m group (p>0.05) was not significant and they did not differ each other in terms of cell proliferation but the cell proliferation in the group of 500-1000 micrometers was better than 150-250 micrometers. (P ⁇ 0.05).
  • cell proliferation in the 1000-500 ⁇ m group was better than 250-500 ⁇ m and in the 250-500 ⁇ m group was better than 150-250 ⁇ m (P ⁇ 0.05).
  • Calvaria tissue is a Hypervascular bone tissue suitable for creating defect in bone and hard tissue regeneration studies and immunogenicity investigation. For this reason, we removed the samples placed inside the rat calvaria after 8 weeks with the aim of histological investigation by hematoxylin and eosin staining. In the control group, which included an empty defect, the repair process of the space created by the soft tissue was observed during the normal process, bone formation stopped at the edge of the defect bone, and no significant signs of new bone were observed.
  • the natural structure of the dentin is visible and the smear layer is clearly distinctive on the structure of the control samples.
  • the mineral structure placed on the surface of the dentin was removed and collagen fibers and the opening of the dentin tubules could be seen free of any mineral crystals.
  • the effect of the day and the effect of the groups together on the cell proliferation rate were statistically significant (p ⁇ 0.05).
  • the groups was observed in the comparison of cell proliferation with each other.
  • the cell proliferation between the Group of 150-250 micrometers compared to the 250-500 micrometers group and the group of 250-500 micrometers compared to the group of 500-1000 micrometers (p> 0.05) was not significant and did not differ in terms of cell proliferation but the 150-250 micrometers group was significant compared to the 250-500 micrometers group (p ⁇ 0.05) and the cell proliferation in the 1000-500 micrometers group was better than the 250-150 micrometers group.
  • cell proliferation p ⁇ o.o5 was significant and all groups were different.
  • LACT Loose fibrous connective tissue
  • FCT fibrous connective tissue
  • R implanted materials
  • the entire defect was filled with the formation of Hypervascular granulation tissue.
  • the remnants of the implanted materials inside the defect were surrounded by giant cells.
  • This invention can be used in all training and laboratory centers as well as dentists

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Abstract

This invention is about the fabrication and evaluation of dentin powder surface modified with alginate on the proliferation of dental pulp stem cells (in vitro) and also the regeneration of dentin organs and immunogenicity (in vivo) root canal treatment of necrosis, traumatized or extended decayed. so we make material to promote dentin-pulp-like tissue to complete the maturation of the apex and thickening wall of the root which degraded during this procedure. the result indicates that surface modification of demineralized dentin powders with alginate is beneficial to improve the mechanical properties of the surface and shows better biocompatibility and minimum immunogenicity which can be used for dentin-pulp-like tissue.

Description

Fabrication and evaluation of dentin powder surface modified with alginate on proliferation of dental pulp stem cells (in vitro) and regeneration of dentin organ and immunogenicity (in vivo)
This invention is about the fabrication and evaluation of dentin powder surface modified with alginate on the proliferation of dental pulp stem cells (in vitro) and also the regeneration of dentin organs and immunogenicity (in vivo) root canal treatment of necrosis, traumatized or extended decayed. so we make material to promote dentin-pulp-like tissue to complete the maturation of the apex and thickening wall of the root which degraded during this procedure. the result indicates that surface modification of demineralized dentin powders with alginate is beneficial to improve the mechanical properties of the surface and shows better biocompatibility and minimum immunogenicity which can be used for dentin-pulp-like tissue.
A61K PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
Medical and dental biomaterial and method of use for the same
United States Patent 8105086
The embodiments herein provide a dental and medical biomaterial and its use for sealing and/or filling the tooth and bone cavities. In the embodiments herein, the calcium salt, calcium oxide, calcium silicate, and calcium phosphate compounds are mixed with a water-based solution, and a bioactive phosphate and calcium-enriched mixture are prepared. The blend comprises a high concentration of water-soluble calcium and phosphate and consequently forms hydroxyapatite during and after setting. The dental/medical biomaterial is biocompatible, antibacterial, and capable of creating an effective seal against the reentrance of microorganisms into the filled cavity. The biomaterial is compatible to handle and set in an aqueous environment and stimulating soft/complex tissue healing/generation/regeneration.
Programming of cells for tolerogenic therapies
United States Patent 9381235
Biomaterial systems, e.g., gel scaffolds, are used in vivo to recruit immune cells and promote their activation towards a non-inflammatory phenotype, thereby leading suppression of inflammation. The compositions and methods are useful to reduce the severity of autoimmunity, chronic inflammation, allergy, and periodontal disease.
Methods for treating dental conditions using tissue scaffolds
United States Patent 7309232
The invention provides methods, apparatus, and kits for regenerating dental tissue in vivo that are useful for treating a variety of dental conditions, exemplified by the treatment of caries. The invention uses tissue scaffold wafers, preferably made of PGA, PLLA, PDLLA or PLGA dimensioned to fit into a hole of the corresponding size drilled into the tooth of the subject to expose dental pulp in vivo. In certain embodiments, the tissue scaffold wafer further comprises calcium phosphate and fluoride. The tissue scaffold wafer may be secured into the hole with a hydrogel, a block of cement, or other suitable material. Either the wafer or the hydrogel or both contain a morphogenic agent, such as a member encoded by the TGF-β supergene family, that promotes regeneration and differentiation of healthy dental tissue in vivo, which in turn leads to remineralization of dentin and enamel. The tissue scaffold may further include an antibiotic or anti-inflammatory agent.
The dental pulp is susceptible to various injuries such as bacteria invasion, caries, and trauma. These factors lead to pulp necrosis, disruption of dentin formation, and prohibition of root development by interfering with the normal physiology of the pulp. The current common clinical treatment attempts to remove pulp necrosis, prevent the inflammation of the apical foramen and partially restore the normal function of the tooth. However, this treatment due to limitations such as difficulty in establishing apical seals in open apex tooth and halting the dentinal wall's development insults to render the walls thin and increase the possibility of tooth fracture. This invention is obtaining a biocompatible and biodegradable biomaterial from bovine dentin under physical and chemical modifications, that can induce the process of tissue regeneration. This can improve the root treatment process through pulp and dentin complex regeneration in the treatment of vital permanent immature open apex teeth. surface-modified dentin powder with alginate, due to the characteristics of this natural polymer such as biocompatibility, biodegradability, and extraordinary plasticity as a suitable scaffold improves dentin-pulp complex regeneration.
The dental pulp is susceptible to various injuries such as bacteria invasion, caries, and trauma. These factors lead to pulp necrosis, disruption of dentin formation, and prohibition of root development by interfering with the normal physiology of the pulp. The current common clinical treatment attempts to remove pulp necrosis, prevent the inflammation of apical foraman and partially restore the normal function of the tooth. However, this treatment due to limitations such as difficulty in establishing apical seals in open apex tooth and halting the dentinal walls development, insults to render the walls thin and increase the possibility of tooth fracture. To solve these problems, the field of regenerative endodontics has presented new possibilities for the treatment of necrotic immature permanent teeth through the development of new pulp tissue based on the meticulous combination and interplay of 3 key elements namely, stem cells, bioactive molecules and scaffolds. One of the effective factors in the regeneration of the dentin-pulp complex is the interaction between stem cells and the dentin matrix. Dentin is a connective tissue, rich in collagen type 1 and bioactive proteins which are release from dentinal tubules under certain conditions, including controlled demineralization. Among these proteins, several growth factors have been found to play a role in tissue repair, cell distinction, and anti-inflammatory processes and it has been proven that these growth factors can be extracted from the surface-modified dentin. This invention is obtaining a biocompatible and biodegradable biomaterial from bovine dentin under physical and chemical modifications, that can induce the process of tissue regeneration. This can improve the root treatment process through pulp and dentin complex regeneration in the treatment of vital permanent immature open apex teeth . surface-modified dentin powder with alginate, due to the characteristics of this natural polymer such as biocompatibility, biodegradability, and extraordinary plasticity as a suitable scaffold improves dentin-pulp complex regeneration.
Solution of problem
One of the effective factors in the regeneration of the dentin-pulp complex is the interaction between stem cells and the dentin matrix. Dentin is an integrated tissue, rich in collagen type 1 and bioactive proteins which are release from dentinal tubules under certain conditions, including controlled demineralization. Among these proteins, several growth factors have been found to play a role in tissue repair, cell distinction, and anti-inflammatory processes and it has been proven that these growth factors can be extracted from surface-modified dentin. Surface-modified dentinal powder with alginate, due to the characteristics of this natural polymer such as biocompatibility, biodegradability, and extraordinary plasticity as a suitable scaffold improves dentin-pulp complex regeneration. However, various studies have been carried out on the use of modified dentin powder but it has been not used in combination with alginate for dentin-pulp complex regeneration and its effect on dental pulp stem cells has not been investigated and according to the information gap in this field, an investigation has been conducted to compare the effect of different sizes of surface-modified bovine dentin powder with alginate on the growth and development of dental pulp stem cells and regeneration of the dentin-pulp structure and immunogenicity of this invention. This study is experimental and its purpose is to investigate the effect of surface-modified bovine dentin powder with alginate in groups of 150-250, 250-500, and 500-1000 micrometers on the proliferation of dental pulp stem cells in laboratory conditions (in vitro) and regeneration of dentin-pulp like structure and immunogenicity in animal (in vivo).
How to conduct research:
-Dentin preparation:
Fresh bovine jaw was harvested from slaughterhouse. Enamel, pulp and periodontal ligament tissue were mechanically removed. Then, the dentin particles were repeatedly washed with distilled water and ethanol 70% ,10 times to remove any contamination from the dentin and were placed under a laminar hood for 24 hours to dry. we ground the dentin particles with a miller and were divided into three sizes of 150-250, 500-250, and 500-1000 micrometers. The samples were washed again with distilled water and ethanol70%,10 times and placed in vacuum oven for 24 hours to dry.
  • Demineralization process:
We demineralized the dentin powders with 37% phosphoric acid for 30 seconds at room temperature, then we washed them with distilled water 5 times to remove residuals. to determine the amount of demineralization in each group, the FE-SEM-EDX test was performed.
  • Atelopeptide procedure:
To remove telopeptides and antigen activity, each mentioned groups was also treated wih pepsin according to the following protocol:
  • The samples were placed in distilled water for 24 hours to wash off any residues of acid.
•Then they were placed in Na2HPO4 0.05 molar solution at pH= 8.5-9 on shaker in cold room (4 c) for 24 hours
•then we washed the samples two times with distilled water, and they were placed in a solution of 0.5 M acetic acid and 0.005 M EDTA on shaker in cold room (4c) for 24 hours.
  • then, 50 mg of 1000 IU pepsin per each gram of dentin powder was added to the current solution and they were placed on the shaker for 24 hours
•Finally, the NaOH was added samples to inactivate pepsin due to reach alkaline pH and washed 4 to 5 times with distilled water.
Surface modification procedure:
After Atelopeptide procedure of dentin powder, the surface modification process was carried out according to the following protocol:
•First, we dissolved the alginate solution containing 5 mg/ml sodium alginate in 0.2 M MES buffer containing 0.3 M NaCl at pH=6.5 on the stirrer.
  • Then we added 0.76% EDC and 0.24% NHS to the alginate solution and placed it on the stirrer for 4 hours.
  • Then we added demineralized atelopeptide dentin powder to this solution and put it on the stirrer for 24 hours.
•Finally, to remove the remnants of alginates that are not attached to the collagens of dentin powders, we washed the samples with PBS regularly. To determine the amount of surface modification in each group, FE-SEM and FTIR tests were performed.
SEM (Scanning Electron Microscopy):
Some of the samples were sputter-coated with a platinum layer of 300 angstroms and were evaluated by SEM (MIRA3TESCAN-XMU) device at 15 kV to examine the dentinal surfaces and tubules.
EDX (energy-dispersive X-ray analysis):
To investigate the mineral structure of dentin more precisely after the demineralization process, we used this test to determine the ratio of calcium to phosphate (mineral structures) and carbon (organic structures) of samples with platinum coating. For this purpose, after evaluating with SEM, the samples were adjusted by the EDX receiver with an accelerating voltage of 20 kV and at a distance of 20 mm. In EDX tests, important parameters include accelerating voltage, scan time, magnification, sample coating, and beam spot size. FTIR-ATR test was used to determine the amount of removal of antigenicity and modification of dentin surface with alginate.
FTIR-ATR test: different wavelengths (infrared) are irradiated to the samples and the amount of absorption of each wavelength is measured, and in this way, the curve of the amount of light absorption according to different wavelengths is obtained for each sample. Considering that amino acids and especially telopeptides have maximum absorption at a specific wavelength, in this way, the number of remaining telopeptides was compared. With this test, we reached the quantitative measurement of the samples.
Investigation of cell proliferation:
This test shows the activity of mitochondria in the cell and has a direct relationship with cell growth and cell survival, which was done according to the protocol of MTT dye. First, a solution of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl 2H-MTT (tetrazolium bromide (Merck, Germany)) was prepared in PBS with a concentration of 5 mg/ml. DPSC cells are planted in 96-well plates (6000 cells per well) in the vicinity of xenograft bovine dentin powder. After 24 hours of incubation, the culture medium containing the tested compounds is changed and replaced with a fresh culture medium containing MTT with a concentration of 0.5 mg/ml, then the cells are placed in an incubator with a temperature of 37 degrees Celsius and 5% CO2 for 3 hours. The insoluble crystals of formazan produced by living cells are dissolved using dimethyl sulfoxide and the quantity of formazan obtained is determined by the spectrophotometric method by a Microplate reader at a wavelength of 570 nm.
Immunogenicity investigation:
All three studied groups were placed inside the defect created in the rat calvaria (skull) to investigate the immunogenicity. Rats were first anesthetized using ketamine xylazine then, in fully sterile conditions, using the scalpel, the long cut was given in rat Calvaria, and using trephine Bur, a defect with a 7mm diameter was created and then the resulting defect was filled with prepared powders and the area was closed with suture and the mice were returned to the maintenance site. After 8 weeks, the rats were sacrificed via anesthesia medicine overdose, and the area under investigation was separated from the skull and placed immediately inside the formalin solution for 2 days. The samples were placed in EDTA for decalcification for 4 weeks and a block was prepared from them using Para film and histological slides were obtained. The obtained slides were stained with H&E stain for histological examination. To examine the histology of the obtained slides, two pathologists examined the slides with an optical microscope. The amount of newly formed bone tissue compared to the total defect created was investigated. To distinguish between newly formed bone with dentin and bone without cells, the indicator of osteocytes inside the lacuna was used. The tissue response adjacent to the implanted samples was carefully examined and ranked in terms of the presence of inflammatory cells, fibrosis, bleeding, necrosis, and the creation of new blood vessels.
To engineer the tissue of the dentin and pulp structure so that eventually creates a tissue similar to the dentin-pulp structure, the use of surface-modified xenograft dentin powder with alginate to improve the structural features of dentin can be a key to solving the limitations of regenerative endodontics. In this study, surface-modified dentin powder with alginate was investigated on the proliferation of dental pulp stem cells and restoration of dentinal structure and immunogenicity. In this study, the dentin powder size index separated the studied groups into three groups: 1. 150-250 micrometers, 2. 250-500 micrometers, and 3. 500-1000 micrometers. The studied groups, that were demineralized by 37% phosphoric acid for 30 seconds, are dentins containing numerous non-collagen proteins that include growth factors such as BMP, as soon as the dentin is demineralized, the dentin tubules become more dilated and can act as a route to flow these basic proteins. To evaluate the quality of demineralization, they were tested with FE-SEM and EDX. Based on the qualitative results obtained, the complete removal of smear layer and smear plug in the opening of dentin tubules and the exposure of collagen fibers and the opening of tubules indicated the success of demineralization in three groups. Since the composition of dentin in terms of volume in the mineral part is 70% by weight and the organic part is 20% and the rest is water, Based on the quantitative results of EDX analysis, the decrease in the weight percentage of calcium and phosphate mineral elements, which are the constituent elements of the mineral structure of dentin, and the increase in the weight percentage of carbon in the organic part, which is one of the constituent elements of the organic structure of dentin, indicated the success of the demineralization process in every Three groups were studied.
Hydroxyapatite crystals, which are one of the basic structures of dentin, can cover the telopeptides of dentinal collagens to prevent their removal in the procedure of pepsin digestion. For this reason, after successful demineralization, the dentin powders were subjected to the Atelopeptide procedure. Then, the present samples were surface modified with alginate according to the mentioned protocol. Specific peaks of the mannuronic acid functional group in wave 884 and uronic acid functional group in wave 939 are specific wavelengths of alginate in the FTIR study. According to the data obtained from all three studied groups and dentin powder compared to alginate, the creation of a peak in the mentioned wavelengths was observed in the groups of surface-modified with alginate. In the qualitative study, the surface-modified samples with alginate compared to the demineralized dentin powder as a control group were photographed. The presence of a uniform layer on the entire outer surface of the dentin and the covering of the exposed collagen fibers and opening of the tubules by alginate compared to the control group indicated the success of the surface modification process.
The general concept of tissue engineering is to combine a matrix or a Scaffold with viable cells to achieve a cell-biomaterial structure to stimulate tissue repair and regeneration by using this structure. Considering the successful production of the surface-modified dentin matrix with alginate in the previous stages, the effect of this structure was investigated in terms of toxicity and cell proliferation on dental pulp stem cells (DPSCs). Based on the toxicity test on days 1, 2, and 3, no toxicity was observed in any of the 3 studied groups, and statistically, the resulting data were not significant compared to each other. (P<0.05) in all groups, cell viability was observed near the dentinal matrix. Also, MTT staining has shown that on the third day, the proliferation of stem cells placed on the dentinal matrix between the 150-250 μm group compared to the 250-500 μm group and also the 250-500 μm group compared to the 500-1000 μm group (p>0.05) was not significant and they did not differ each other in terms of cell proliferation but the cell proliferation in the group of 500-1000 micrometers was better than 150-250 micrometers. (P<0.05). On the seventh day and the fourteenth day, cell proliferation in the 1000-500 µm group was better than 250-500 µm and in the 250-500 µm group was better than 150-250 µm (P<0.05).
Finally, in all groups, cell proliferation was on day 14 more than day 7 and on day 7 more than day 3. After completing the laboratory phase, to investigate immunogenicity and tissue reaction, we placed all 3 studied groups in the animal phase inside the rat calvari. Calvaria tissue is a Hypervascular bone tissue suitable for creating defect in bone and hard tissue regeneration studies and immunogenicity investigation. For this reason, we removed the samples placed inside the rat calvaria after 8 weeks with the aim of histological investigation by hematoxylin and eosin staining. In the control group, which included an empty defect, the repair process of the space created by the soft tissue was observed during the normal process, bone formation stopped at the edge of the defect bone, and no significant signs of new bone were observed. In the center part, Loose fibrous connective tissue and granulation tissue were clearly observed. The minimum immunogenicity and inflammation was observed in the 500-1000 group and then in the 250-500 group. In the 150-250 group, more inflammatory reactions were reported than the other two groups, but this reaction was at the level of the control group. Therefore, all the studied groups had acceptable immunogenicity and biocompatibility in close proximity to the tissue, and no significant and acute immunogenicity was reported in any of them. As a routine process, the body's innate immunity system shows a more acute immune response when faces with nano-sized substances than micro-sized substances through the secretion of IL-β1 cytokines and the calling of immune cells.
In terms of observation of hard tissue in the 500-1000 micrometers group, the formation of newly-formed bone tissue was more comparable than other groups and the amount of fibrous tissue was significantly lower than other groups. Also, the evolution of the newly-formed bone tissue in this group was more clear, although most of the defect space was filled with fibrous tissue and a high angiogenesis was clearly observed, statistically, this group has shown significant results compared to the other two groups (P<0.05) the speed of making new bone tissue was more than the speed of dentinal matrix resorbtion in this group, however, it was opposite in smaller groups and the speed of the samples resorbtion were fast since the time required to induce ossification and for this reason, in other groups, instead of hard tissue, we have the invasion of connective tissue into the space. The amount of angiogenesis in the groups of 250-500 micrometers and 500-1000 micrometers was more comparable than the control group. Finally, all the results obtained from the implanted groups have shown acceptable biocompatibility. Surface-modified dentin powder with alginate with a positive effect on the proliferation of stem cells and fail to create immunogenicity can be investigated as a suitable material for regeneration of the dentin-pulp like structure.
Advantage effects of invention
1) preserving the life of the dental pulp
2) sustaining strength and helping to increase the thickness of tooth dentin
3) Reinforcement and improvement of the strength of immature open apex teeth that requires root treatment
4) Areas for Improvement in performing non-invasive root canal treatment
5) Biocompatibility and biodegradability of the material
6) loaded of growth factors in the material
7) Lack of toxicity in close proximity to stem cells
8) Positive effect on the proliferation of stem cells
9) Favorable response of immunogenicity and biocompatibility in animal test
10) Using the biomaterial regeneration potentials instead of industrial materials
11) Innovative treatment method compared to the common traditional methods
: schematic of working plan
: schematic of working plan
Examples
The findings and results:
Investigating the demineralization of dentinal powders: (table 1)
According to the obtained FE-SEM images in three magnifications of the control samples in the sizes of 150-250 micrometers, 250-500 micrometers, and 500-1000 micrometers, the natural structure of the dentin is visible and the smear layer is clearly distinctive on the structure of the control samples. The samples demineralized by 37% phosphoric acid, in the sizes of 150-250 micrometers, 250-500 micrometers, and 500-1000 micrometers, clearly have shown the removal of the smear layer and smear plugs of the opening of dentin tubules. The mineral structure placed on the surface of the dentin was removed and collagen fibers and the opening of the dentin tubules could be seen free of any mineral crystals. In the results of the EDX test to investigate the changes in the weight percentage of calcium and phosphor mineral elements except for organic carbon, between the control samples and the demineralized samples, the decrease in the weight percentage of calcium and phosphor elements except organic carbon in all demineralized groups compared to the control was seen.
[Table .1]: Investigating the demineralization of dentinal powders
Investigating the surface modification process with alginate:
According to the study of three size groups of surface-modified dentin powder with alginate and demineralized dentin powder as a control group compared to the peaks of alginate powder, at the wavelength of 884 to 939, which is the exclusive wavelengths of the Functional group of monomeric acid and uronic acid of alginate. , the peak change in all three groups compared to the control group has been created. In the comparison of the graph of recorded waves from the surface-modified three size groups, the 500-1000 micrometers group compared to the 250-500 micrometers group and the 250-500 micrometers group compared to the 150-250 micrometers group had better surface modification (table 2).
[Table .2]: Investigating the surface modification process with alginate
In examining the FE-SEM images obtained from the surface-modified samples with alginate compared to the demineralized dentin powder samples, qualitatively the presence of a thin layer of alginate on the collagen fibers, that were previously exposed by 37% phosphoric acid, and in the opening of the dentin tubules can be seen (table 3).
[Table .3]
Investigation of cytotoxicity (table 4):
Based on the results of the cell viability graph obtained from the MTT test on days 1, 2, and 3 in the cell culture medium, it was shown that none of the three size groups of surface-modified powder with alginate were toxic. According to the statistical analysis, (P>0.05) the data of the groups were not significant compared to each other and all the groups showed the same level of cell viability.
[Table .4]: Investigation of cytotoxicity
Investigation of cell proliferation (table 5):
The effect of the day and the effect of the groups together on the cell proliferation rate were statistically significant (p<0.05). On the third day (p <0.05) the groups was observed in the comparison of cell proliferation with each other. The cell proliferation between the Group of 150-250 micrometers compared to the 250-500 micrometers group and the group of 250-500 micrometers compared to the group of 500-1000 micrometers (p> 0.05) was not significant and did not differ in terms of cell proliferation but the 150-250 micrometers group was significant compared to the 250-500 micrometers group (p<0.05) and the cell proliferation in the 1000-500 micrometers group was better than the 250-150 micrometers group. On the seventh and fourteenth day, cell proliferation (p <o.o5) was significant and all groups were different. Cell proliferation in the 500-1000 micrometers group was better than 250-500 micrometers and the 250-500 micrometers group was better than 150-250 micrometers. In the comparison of each group only on days 3, 7, and 14, according to the diagram, it was observed that in all groups, cell proliferation was more on day 14 than day 7 and on day 7 more than day 3.
Table 5: Investigation of cell proliferation
Investigation of immunogenicity and tissue reaction:
Control group (empty defect):(table 6)
The entire defect was covered with Loose fibrous connective tissue (LACT). No significant signs of new bone formation were observed and the granulation tissue formed inside the defect was significant.
150-250 micrometers group:
The entire defect was simultaneously covered with the fibrous connective tissue (FCT) and the remains of the implanted materials (R). Not only the formation of newly formed bone (NB) was insignificant, but also the remnants of the implanted materials were removed from the defect site by multinucleated giant cells and filled the created space with fibrous connective tissue.
250-500 micrometers group:
The entire defect was filled with the formation of Hypervascular granulation tissue. The remnants of the implanted materials inside the defect were surrounded by giant cells.
500-1000 micrometers group:
In this group, the formation of newly formed bone tissue was more compared to other groups, and the amount of fibrous tissue was significantly lower than other groups. Also, the development of newly formed bone tissue was more clear in this group. Although most of the defect space was filled with fibrous tissue and a lot of angiogenesis was clearly observed, statistically this group has shown significant results compared to the other two groups (P<0.05). The amount of angiogenesis in the 250-500 µm and 500-1000 µm groups is higher than the control group. Finally, all the results obtained from the implanted groups have shown acceptable biocompatibility.
[Table .6]: Investigation of immunogenicity and tissue reaction
This invention can be used in all training and laboratory centers as well as dentists

Claims (5)

  1. This invention is about the fabrication and evaluation of dentin powder surface modified with alginate on the proliferation of dental pulp stem cells (in vitro) and also the regeneration of dentin organs and immunogenicity (in vivo) root canal treatment of necrosis, traumatized or extende
  2. d decayed. so we make material to promote dentin-pulp-like tissue to complete the maturation of the apex and thickening wall of the root which degraded during this procedure.
  3. according to to claim 1, the methods and materials are dentin powders in 3 groups (1-150-250μ 2-250-500μ 3-500-1000μ) after demineralization and telopeptide procedure, were grafted with alginate and evaluated for FE-SEM, EDX, FTIR, cell viability and proliferation of human dental pulp stem cells (DPSCs) and then all the groups were settled in 6mm defect of rat’s calvaria for 8 weeks to assess immunogenicity.
  4. all the groups had successful results for demineralization and telopeptide procedure. Cell viability results were not significant but cell proliferation in 500-1000μ was significantly higher than others (p<0.05). immunological response in all groups was minimum but in 500-1000 was the least.
  5. the result indicates that surface modification of demineralized dentin powders with alginate is beneficial to improve the mechanical properties of the surface and shows better biocompatibility and minimum immunogenicity which can be used for dentin-pulp-like tissue.
PCT/IB2022/062774 2022-12-25 2022-12-25 Fabrication and evaluation of dentin powder surface modified with alginate on proliferation of dental pulp stem cells (in vitro) and regeneration of dentin organ and immunogenicity (in vivo) WO2023135481A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006116530A2 (en) * 2005-04-28 2006-11-02 The Trustees Of Columbia University In The City Of New York Compositions and methods for treating pulp inflammations caused by infection or trauma

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006116530A2 (en) * 2005-04-28 2006-11-02 The Trustees Of Columbia University In The City Of New York Compositions and methods for treating pulp inflammations caused by infection or trauma

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ATHIRASALA AVATHAMSA, TAHAYERI ANTHONY, THRIVIKRAMAN GREESHMA, FRANÇA CRISTIANE M, MONTEIRO NELSON, TRAN VICTOR, FERRACANE JACK, B: "A dentin-derived hydrogel bioink for 3D bioprinting of cell laden scaffolds for regenerative dentistry", BIOFABRICATION, vol. 10, no. 2, pages 024101, XP093080629, DOI: 10.1088/1758-5090/aa9b4e *
BAKHTIAR HENGAMEH; MAZIDI AMIR; MOHAMMADI-ASL SAEED; HASANNIA SADEGH; ELLINI MOHAMMAD REZA; PEZESHKI-MODARESS MOHAMMAD; OSTAD SEYE: "Potential of Treated Dentin Matrix Xenograft for Dentin-Pulp Tissue Engineering", JOURNAL OF ENDODONTICS, ELSEVIER, AMSTERDAM, NL, vol. 46, no. 1, 20 November 2019 (2019-11-20), AMSTERDAM, NL , pages 57, XP085947498, ISSN: 0099-2399, DOI: 10.1016/j.joen.2019.10.005 *
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