Indigenous Bioactive Dental Cement Mineral Pentoxide Aggregate

Abstract

ABSTRACT Indigenous bioactive dental cement Mineral Pentoxide Aggregate Regenerative Endodontics relies on the triad of stem cells, scaffolds and growth factors for an effective outcome. This regenerative process is important in returning the periapical tissues to their original status after root canal treatment. Hence regeneration through cell homing techniques require that chemotactic bioactive materials are used appropriately. In our quest to find the ideal bioactive material in Endodontics, we have experimentally developed an indigenous bioactive cement that has many properties like the contemporary calcium silicate cements. Preliminary investigations revealed that they set faster and discolour less than contemporary calcium silicate cements and demonstrated equivalent biocompatibility. Bioactivity was assessed by gene marker expression of dentin sialophosphoprotein (DSPP) and vascular endothelial growth factor (VEGF) with and without scaffolds and found to be superior for the indigenous bioactive material

Specification

Description:
FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
Complete Specification
(See section 10 and rule 13)


1.Title of the Invention : Indigenous bioactive dental cement Mineral Pentoxide Aggregate


2. Applicant
Name Nationality Address


Dr. Anand V. Susila




Indian



Professor and HOD, Department of Conservative dentistry and Endodontics, Madha Dental College, Somangalam Road, - Chennai 600 069



3. Preamble to the Description :

The following specification particularly describes the invention and the manner in which it is to be performed.

4. DESCRIPTION
Field of the Invention
The present invention relates to the field of Dentistry.
Background of the Invention
Regenerative Endodontics relies on the triad of stem cells, scaffolds and growth factors for an effective outcome. This regenerative process is important in returning the periapical tissues to their original status after root canal treatment. The stimulation of growth factors and chemotaxis of stem cells is facilitated by bio active materials like calcium silicate cements.1-5
Calcium silicate cements introduced as a re-incarnation of Portland cement offer excellent biocompatibility, bioactivity and sealing which are also essential in a root canal treatment. There are many different types of commercially available calcium silicate cements which are also known as hydraulic cements, tri calcium silicate cements, bioceramics so on and so forth. There are meta analytical data available in the literature supporting their beneficial properties in tissue regeneration. A systematic review by Rathinam et al. found that various biochemical and gene markers for regeneration like alkaline phosphatase (ALP), collagen I (COL1), bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), Runt related transcription factor 2 (RunX2) were expressed1 in all three cell types like osteoblasts, cementoblasts and odontoblasts by different types of calcium silicate cements like ProRoot MTA, Biodentin, Bioaggregate, calcium silicate, MTA Angelus, Endosequence root repair material and MTA Plus.
MTA has been shown to activate all three kinase pathways of mitogen activated protein kinase (MAPK) namely p38, ERK1/2 and JNK1/2/3 by intra cellular Ca signalling in stem cells leading to their differentiation into osteoblast and odontoblast lineages.1 Hence regeneration through cell homing techniques require that chemotactic bioactive materials are used appropriately.6-10 However, there are few disadvantages like high cost, slow setting, discoloration of tooth and gingiva.
Scaffolds are essential for voluminous regenerative work.10,11 The synergistic functioning of scaffolds and bioactive materials cannot be overemphasized in this context. If this synergism is either achieved or accentuated then regeneration is just a matter of time for nature to complete. Platelet based scaffolds like platelet rich plasma (PRP) and fibrin (PRF) are easy to obtain, autologous and have excellent regenerative potential according to several studies.10-13
In our quest to the ideal bioactive material in Endodontics, we have experimentally developed an indigenous bioactive cement that has many properties like the contemporary calcium silicate cements. Preliminary investigations revealed that they set faster and discolour less than contemporary calcium silicate cements and demonstrated equivalent biocompatibility. Bioactivity was assessed by gene marker expression of dentin sialophosphoprotein (DSPP) and vascular endothelial growth factor (VEGF) with and without scaffolds and found to be superior for the indigenous bioactive material.
Summary of the invention
Regenerative Endodontics relies on the triad of stem cells, scaffolds and growth factors for an effective outcome. This regenerative process is important in returning the periapical tissues to their original status after root canal treatment. The stimulation of growth factors and chemotaxis of stem cells is facilitated by bio active materials like calcium silicate cements. Calcium silicate cements introduced as a re-incarnation of Portland cement offer excellent biocompatibility, bioactivity and sealing which are also essential in a root canal treatment. There are many different types of commercially available calcium silicate cements which are also known as hydraulic cements, tri calcium silicate cements, bioceramics so on and so forth. A systematic review by Rathinam et al. found that various biochemical and gene markers for regeneration like alkaline phosphatase (ALP), collagen I (COL1), bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), Runt related transcription factor 2 (RunX2) were expressed1 in all three cell types like osteoblasts, cementoblasts and odontoblasts by different types of calcium silicate cements like ProRoot MTA, Biodentin, Bioaggregate, calcium silicate, MTA Angelus, Endosequence root repair material and MTA Plus. MTA has been shown to activate all three kinase pathways of mitogen activated protein kinase (MAPK) namely p38, ERK1/2 and JNK1/2/3 by intra cellular Ca signalling in stem cells leading to their differentiation into osteoblast and odontoblast lineages. Hence regeneration through cell homing techniques require that chemotactic bioactive materials are used appropriately. However, there are few disadvantages like high cost, slow setting, discoloration of tooth and gingiva. The synergism between scaffolds and bioactive materials used in regenerative endodontic procedures (REPs) is worth investigation to maximize their potential and have predictable treatment outcome. If this synergism is either achieved or accentuated then regeneration is just a matter of time for nature to complete. Platelet based scaffolds like platelet rich plasma (PRP) and fibrin (PRF) are easy to obtain, autologous and have excellent regenerative potential according to several studies. In our quest to the ideal bioactive material in Endodontics, we have experimentally developed an indigenous bioactive cement that has many properties like the contemporary calcium silicate cements. Preliminary investigations revealed that they set faster and discolour less than contemporary calcium silicate cements and demonstrated equivalent biocompatibility. Bioactivity was assessed by gene marker expression of dentin sialophosphoprotein (DSPP) and vascular endothelial growth factor (VEGF) with and without scaffolds and found to be superior for the indigenous bioactive material.
Detailed description of the invention:
A novel indigenous bioactive cement Mineral Pentoxide Aggregate was formulated with the help of sol-gel process using calcium oxide, silicon dioxide, strontium oxide, zirconium dioxide, zinc oxide, calcium hydroxide, calcium chloride and strontium chloride. After drying the gel, the resultant particles were milled to obtain nano sized cement particles.
Experiments were conducted to check the setting time, colour stability, biocompatibility and bioactivity.
Setting time:
Experiments were done according to ISO-6876. MTA Angelus (Angelus, PR, Brazil) was used as the control group. Both the indigenous novel bioactive cement Mineral Pentoxide Aggregate and the control MTA Angelus were mixed in the powder liquid ratio of 3:1. The indigenous novel bioactive cement Mineral Pentoxide Aggregate was mixed with distilled water, while MTA was mixed with the liquid (distilled water) provided by the manufacturer. Stainless steel moulds measuring 5mm in diameter and 2mm in height were used to make 10 numbers of disc shaped samples of each cement. A Gilmore needle weighing 100g with a tip diameter of 2mm was used to indent the cement samples for a duration of 3s, 3min after the start of mixing time. This was continued at an interval of 1min till cement was not adhering to the tip (initial setting time) and no indentation was made on the surface (final setting time).
 Colour stability:
Moulds similar to those used for setting time were used to prepare samples (10) of the same control group MTA and indigenous novel bioactive cement Mineral Pentoxide Aggregate after mixing in the same ratio as mentioned for setting time experiment. The cements were allowed to mature completely to reach their full mechanical properties under 37 0C at 100% humidity for 24h. After this maturation the baseline L*, a*, b* values were measured using Vita Easy Shade Spectrophotometer under white ambient light. Triplicate values were recorded for each sample and the average taken. Three aging and discolouring solutions were used to test their colour stability. Distilled water, 5.25% sodium hypochlorite and human blood from healthy consenting volunteer was used. The samples were immersed in the respective solutions for 3days. For distilled water and sodium hypochlorite, experiment was continued till 28days duration thereafter. The L*, a*, b* values were measured similar to previous explanation at the end of each time period. The ΔE values were calculated by taking the baseline values as standard for each cement
Biocompatibility:
Caries-free extracted human teeth were split in sagittal direction with a mechanised chisel and the pulp tissue salvaged for cultivating human dental pulp stem cells. It was immersed in phosphate buffered saline. Collagenase Type I at 0.1% was added to the tissue and digestion allowed for 30min. The resultant cell suspension was grown in Dulbecco's modified Eagle's medium with 20% foetal bovine serum, 1% penicillin (10000 U/mL) and streptomycin (10000 mg/mL) in a humidified atmosphere at 37 0C and 5% CO2. The media was renewed every 3 days and three passages were done before harvesting the cells for experiments. The stemness of the cells was confirmed with microscopic examination, osteo, adipo and chondrogenic differentiation at 4 weeks.
Ten samples of each material (MTA Angelus and indigenous novel bioactive cement Mineral Pentoxide Aggregate) measuring 1mg/mL were immersed in Dulbecco's modified Eagle's medium without foetal bovine serum for 24h and 3 days. After this immersion period the media were collected and treated with cells for testing biocompatibility.
Each cell culture well received 500 µL of test sample present in the immersed media. After culturing for 24h and 72h, 10 µL/100mL of MTT reagent (5mg/mL stock) was added and incubated for 4h. Formazan dye formation was allowed at 37 0C and the media exchanged for 200 µL DMSO. The cultures were allowed to stand for 10min. The reaction product was transferred to 96 well ELISA plate and cell viability quantified using a A590 ELISA plate reader.
Bioactivity:
Preparation of PRF
Fresh blood of 3ml volume was collected in 10 ml test tubes without anticoagulant from consenting healthy adult human volunteers. PRF was obtained by centrifuging at 1500 rpm for 10 min and compressed in sterile gauze pad to obtain as a membrane14 which was cut into required portions and the subsequent experiments conducted.
For sample preparation for the groups with scaffolds, a powder:liquid of 1:1 for both MTA Angelus and indigenous novel bioactive cement Mineral Pentoxide Aggregate was used to enable mixing with PRF. PRF was cut into 3mm square pieces and used. For sample preparation for the groups without scaffolds, a powder:liquid of 3:1 was used for both materials. The samples were stored in sterile test tubes for 24h at 20 0C before adding to the cells.
A stock solution of 1 mg/mL test samples was prepared in media containing alpha MEM supplemented with penicillin (10000 U/mL) and streptomycin (10000 mg/mL). The treatment media was prepared by adding 50 μl of stock solution to 1ml of differentiation media containing alpha MEM supplemented with 10 mM β-glycerophosphate and 0.05 mM Ascorbic acid.
Using a quantitative real-time PCR technique, the impact of the materials on the expression of osteogenic and angiogenic marker genes was examined. For 14 days, the human dentin pulp stem cells were exposed to treatment media, which promotes osteogenic differentiation in the cells. Using oligo (dT), total RNA was reverse transcribed into cDNA after being extracted using the Trizol reagent. SYBR Green Supermix, cDNA, and specific primers for each gene were combined in a 25-μl combination to produce quantitative real-time PCR experiments. The double-stranded DNA binding dye SYBR Green's fluorescence allowed for the detection of the particular PCR products. Using the comparison Ct value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the threshold cycle (Ct) value of each PCR product was obtained. This was used to compute the relative mRNA expression levels. All experiments were performed in triplicate and repeated at least three times. The primer sequences are shown in Table 1.
Statistical Analysis:
ANOVA, post-hoc Tukey's and paired t-tests were done to analyse the statistical significance of the experiments at a significance level of p≤0.05 and 95% confidence interval.
Results:
Setting time:
The indigenous novel bioactive cement Mineral Pentoxide Aggregate had an initial setting time of 7.6min, which was statistically significantly lower (p= 2.25E-12) than that of MTA Angelus at 61.8min. The final setting time of indigenous novel bioactive cement Mineral Pentoxide Aggregate was 24.2min which was also statistically significantly lower (p=2.7E-11) than that of MTA Angelus at 119.6 min.
Colour Stability:
The indigenous novel bioactive cement Mineral Pentoxide Aggregate had a ΔE value not significantly different (p=0.7) from baseline at 3 days for all three aging solutions namely distilled water, sodium hypochlorite and blood. MTA Angelus also had a ΔE value not significantly different (p=0.6) from baseline at 3 days for distilled water. However, it had higher (p=0.018) ΔE values for sodium hypochlorite and blood at 3 days than baseline.
On comparing the intergroup ΔE values at 3 days, the indigenous novel bioactive cement Mineral Pentoxide Aggregate was not significantly different from MTA in distilled water (p=0.5) but showed significantly less discolouration and lower ΔE values in sodium hypochlorite (p=0.004) and blood (p=0.0002).
At 28 days, there was significant (p=0.03) colour change in all three solutions for both the cements. But the indigenous novel bioactive cement Mineral Pentoxide Aggregate was significantly more (p=0.0001) colour stable by exhibiting lower ΔE value than MTA Angelus in both sodium hypochlorite and blood. They showed statistically insignificant difference of (p=0.3) ΔE value in distilled water. The differences were observed in both comparison: baseline vs. 28 days and 3 vs. 28 days.
Biocompatibility:
Both the materials exhibited similar biocompatibility in human dental pulp stem cells (68.7% cell viability - indigenous novel bioactive cement Mineral Pentoxide Aggregate; 71.4% - MTA Angelus) at 24h with no statistically significant difference (p=0.94). At 3 days (72h), also their biocompatibility was very similar (67.1% cell viability - indigenous novel bioactive cement Mineral Pentoxide Aggregate; 67.7% - MTA Angelus) with no statistically significant difference (p=0.99).
Bioactivity:
Both DSPP and VEGF were significantly more expressed (p=0.002) by indigenous novel bioactive cement Mineral Pentoxide Aggregate than MTA Angelus. There was a significantly greater expression of VEGF by indigenous novel bioactive cement Mineral Pentoxide Aggregate than MTA Angelus (p=0.0003). The indigenous novel bioactive cement Mineral Pentoxide expressed both markers similar to PRF (p=0.7).
In the presence of scaffold, both materials better expressed both the gene markers DSPP and VEGF than alone (p=0.03). The indigenous novel bioactive cement Mineral Pentoxide expressed significantly more marker genes for both DSPP and VEGF in the presence of PRF (p=0.01) than MTA Angelus.
References:
1. Rathinam E, Rajasekharan S, Chitturi RT, Declercq H, Martens L, De Coster P. Gene Expression Profiling and Molecular Signaling of Various Cells in Response to Tricalcium Silicate Cements: A Systematic Review. Journal of Endodontics. 2016;42(12):1713-1725. doi:10.1016/j.joen.2016.08.027
2. Chung M, Lee S, Chen D, et al. Effects of Different Calcium Silicate Cements on the Inflammatory Response and Odontogenic Differentiation of Lipopolysaccharide-Stimulated Human Dental Pulp Stem Cells. Materials. 2019;12(8):1259. doi:10.3390/ma12081259
3. Maru V, Dixit U, Shetty A. Biocompatibility, Bioactivity and Gene Expression Analysis of SHEDS Cultured in Various Calcium Silicate Based Cements: A Systematic Review and Meta-Analysis of in Vitro Studies. Journal of Clinical Pediatric Dentistry. 2022;46(3):171-182. doi:10.17796/1053-4625-46.3.1
4. Assadian H, Khojasteh A, Ebrahimian Z, et al. Comparative evaluation of the effects of three hydraulic calcium silicate cements on odontoblastic differentiation of human dental pulp stem cells: an in vitro study. J Appl Oral Sci. 2022;30:e20220203. doi:10.1590/1678-7757-2022-0203
5. Manriquez-Olmos L, Garrocho-Rangel A, Pozos-Guillén A, Ortiz-Magdaleno M, Escobar-García DM. Effect of tricalcium silicate cements in gene expression of COL1A1, MAPK's, and NF-kB, and cell adhesion in primary teeth' pulp fibroblasts. JOCPD. 2022;46(6):17. doi:10.22514/jocpd.2022.021
6. Saoud TM, Martin G, Chen YHM, et al. Treatment of Mature Permanent Teeth with Necrotic Pulps and Apical Periodontitis Using Regenerative Endodontic Procedures: A Case Series. Journal of Endodontics. 2016;42(1):57-65. doi:10.1016/j.joen.2015.09.015
7. Jiang X, Liu H, Peng C. Clinical and Radiographic Assessment of the Efficacy of a Collagen Membrane in Regenerative Endodontics: A Randomized, Controlled Clinical Trial. Journal of Endodontics. 2017;43(9):1465-1471. doi:10.1016/j.joen.2017.04.011
8. Lin J, Zeng Q, Wei X, et al. Regenerative Endodontics Versus Apexification in Immature Permanent Teeth with Apical Periodontitis: A Prospective Randomized Controlled Study. Journal of Endodontics. 2017;43(11):1821-1827. doi:10.1016/j.joen.2017.06.023
9. Meschi N, EzEldeen M, Torres Garcia AE, Jacobs R, Lambrechts P. A Retrospective Case Series in Regenerative Endodontics: Trend Analysis Based on Clinical Evaluation and 2- and 3-dimensional Radiology. Journal of Endodontics. 2018;44(10):1517-1525. doi:10.1016/j.joen.2018.06.015
10. Nageh M, Ahmed GM, El-Baz AA. Assessment of Regaining Pulp Sensibility in Mature Necrotic Teeth Using a Modified Revascularization Technique with Platelet-rich Fibrin: A Clinical Study. Journal of Endodontics. 2018;44(10):1526-1533. doi:10.1016/j.joen.2018.06.014
11. Shivashankar VY. Comparison of the Effect of PRP, PRF and Induced Bleeding in the Revascularization of Teeth with Necrotic Pulp and Open Apex: A Triple Blind Randomized Clinical Trial. JCDR. Published online 2017. doi:10.7860/JCDR/2017/22352.10056
12. Yan H, De Deus G, Kristoffersen IM, et al. Regenerative Endodontics by Cell Homing: A Review of Recent Clinical trials. Journal of Endodontics. 2023;49(1):4-17. doi:10.1016/j.joen.2022.09.008
13. Rahul M, Lokade A, Tewari N, et al. Effect of Intracanal Scaffolds on the Success Outcomes of Regenerative Endodontic Therapy - A Systematic Review and Network Meta-analysis. Journal of Endodontics. 2023;49(2):110-128. doi:10.1016/j.joen.2022.11.011
14. Palaiologou A, Keeling F. Autologous blood products: Usage and preparation protocols. Clin Adv Periodontics. 2022;12(4):287-293. doi:10.1002/cap.10221
15. Taihi I, Pilon C, Cohen J, et al. Efficient isolation of human gingival stem cells in a new serum-free medium supplemented with platelet lysate and growth hormone for osteogenic differentiation enhancement. Stem Cell Res Ther. 2022;13(1):125. doi:10.1186/s13287-022-02790-7
16. Huang CK, Huang W, Zuk P, et al. Genetic Markers of Osteogenesis and Angiogenesis Are Altered in Processed Lipoaspirate Cells when Cultured on Three-Dimensional Scaffolds: Plastic and Reconstructive Surgery. 2008;121(2):411-423. doi:10.1097/01.prs.0000298510.03226.5f
17. Chen CH, Lin YS, Fu YC, et al. Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro. Journal of Applied Physiology. 2013;114(5):647-655. doi:10.1152/japplphysiol.01216.2012.
 
 
 

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, Claims:CLAIMS
I Claim:
1. A bioactive cement Mineral Pentoxide Aggregate formulated with the help of sol-gel process using calcium oxide, silicon dioxide, strontium oxide, zirconium dioxide, zinc oxide, calcium hydroxide, calcium chloride and strontium chloride, wherein after drying the gel, the resultant particles were milled to obtain nano sized cement particles.
2. The bioactive cement Mineral Pentoxide Aggregate as claimed in claim 1, wherein Nano particulate bioactive cement for dentistry sets by hydration reaction.
3. The bioactive cement Mineral Pentoxide Aggregate as claimed in claim 1, wherein the bioactive cement for regenerative dentistry is Fast setting, colour stable and indigenous.
4. The bioactive cement Mineral Pentoxide Aggregate as claimed in claim 1, wherein the bioactive cement for conservative dentistry and endodontics applications has enhanced angiogenic potential.
5. The bioactive cement Mineral Pentoxide Aggregate as claimed in claim 1, wherein, the bioactive cement useful in revascularization.
6. The bioactive cement Mineral Pentoxide Aggregate as claimed in claim 1, wherein the bioactive cement is with comparable biocompatibility to commercially available MTA.
Dated this the 14th November 2024

Senthil Kumar B
Agent for the applicant
IN/PA-1549

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