AU2021102741A4 - A novel methodology for the production and inoculation of periodontal ligament stem cells from periodontal ligament fibers on titanium implants in an animal model - Google Patents
A novel methodology for the production and inoculation of periodontal ligament stem cells from periodontal ligament fibers on titanium implants in an animal model Download PDFInfo
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- 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
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- C12N2500/84—Undefined extracts from animals from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
Human tissues have been designed uniquely. In the current times, there has arisen another and
aggressive approach that joins information from material science with cell science and
medication. These are called realms of tissue engineering science. These techniques have utilized
biodegradable polymers to make frameworks into the cells, embedded to create tissues within the
sight of development factors. In a human body a single embryonic stem cell can be differentiate
into specialized 220 cell types. However, embryonic stem cells Introduction has a high potential
for causing tumours; making them less desirable to use than adult stem cells. With effective
minimally invasive procedures, the progenitor cells in periodontal tissue has an extensive version
as vitro, offers a singular pool of stem cells. They are a viable alternative to bone marrow
aspiration, as procurement of PDLSCs from premolars is the less invasive and morbid procedure
with minimal complications. The present study findings suggest that PDLSCs can be isolated and
cultured from periodontal ligament cells of extracted adult premolars by using CD45, CD73,
CD90, CD105 and CD146 markers. The current work elicits that CD 146, CD 105, CD 90, CD
73 is a reliable positive marker for the identification of periodontal ligament stem cells and CD
45 is a reliable negative marker for the identification of periodontal ligament stem cells.
Periodontal formation occurs during cultured PDLSCs on the dedicated surface of titanium,
which can be confirmed both histologically and radiologically. It can safely be postulated that
from the present study that tissue engineering of the PDL has been achieved as a proof-of
concept in present study. We have observed important intertissued interactions like the formation
of a functional PDL around the implantation site, and induction of the formation of bone in the
vicinity of the implants. Ligaplants as tooth substitutions may have unequivocal focal points as
contrasted and osseointegrated gadgets, because of their potential for periodontal tissue recovery.
It is recommended that restorative achievement requires the capacity of a high extent of the
developed cells to coordinate into another PDL. The ligaplant medical procedure is moderately
simple because the embed isn't firmly fitted into its site. Future clinical utilization of ligaplants
may stay away from bone uniting with its cost, bother, and uneasiness for the patient. The
bioengineering of clinical implants of PDL would be tremendous alternative for stable and
natural tooth implant therapy.
1
Sample Images
Figure-4: Positive Staining for CD90 (20X
Figure-i:Scraping ofPDLcellsfromextracted magnification)
teeth
B C
Figure-2: Colonies of PDLSCs formed from Figure-5: Positive Staining for CD105 (20X
isolated PDLSCs at 14 days magnification)
D
Figure-3: Positive Staining for CD73 (20X Figure-6: Positive Staining for CD146 (20X
magnification) magnification)
1
Description
Sample Images
Figure-4: Positive Staining for CD90 (20X Figure-i:Scraping ofPDLcellsfromextracted magnification) teeth
Figure-2: Colonies of PDLSCs formed from Figure-5: Positive Staining for CD105 (20X isolated PDLSCs at 14 days magnification)
Figure-3: Positive Staining for CD73 (20X Figure-6: Positive Staining for CD146 (20X magnification) magnification)
Title: A novel methodology for the production and inoculation of periodontal ligament stem cells from periodontal ligament fibers on titanium implants in an animal model
1. General introduction Though the field of regenerative dentistry is in the process of significant modifications and adequate advancements, the implant inserts are being put with the point of achieving osseointegration without offering any thought to the recovery of periodontium around the embedded bony areas. Human tissues have been designed uniquely. In the current times, there has arisen another and aggressive approach that joins information from material science with cell science and medication. These are called realms of tissue engineering science. These techniques have utilized biodegradable polymers to make frameworks of the desired cells, embedded to create tissues within the sight of development factors. In a human body, a single embryonic stem cell can be differentiated into specialized 220 cell types. However, embryonic stem cells Introduction has a high potential for causing tumours; making them less desirable to use than adult stem cells.
2. Objective/objectives of the work The main objective of this work suggest that PDLSCs can be isolated and cultured from periodontal ligament cells of extracted adult premolars by using CD45, CD73, CD90, CD105 and CD146 markers. The current work elicits that CD 146, CD 105, CD 90, CD 73 is a reliable positive marker for the identification of periodontal ligament stem cells and CD 45 is a reliable negative marker for the identification of periodontal ligament stem cells.
3. Detailed description of work Ligaplants are purely unique. The PDL, when it is attached to the structural framework of the exterior implant biomaterial, it forms a new entity termed a Ligaplant. Not very long ago, lost teeth without considering the PDL into account were replaced. The end useable implants of an ideal biomaterial are straightforwardly embedded into jawbones. Before these strategies, all neighbourhood bone deformities and by and large all the hapless bone quality regions require bone reconstruction. Besides that, confined bone misalignment around the embed installation speaks of a clinical challenge. It is particularly true in the instances of gingival deficiency where, perhaps because of altered tissue engineering, further careful interventions are warranted. So, to conquer this issue, an embedded framework with tissue-actuating properties may be valuable. Inserts with PDL might be introduced in the extraction attachment of the missing tooth. This is a unique way. It encourages implant surgery. It can safely be postulated that from the present study. Ligaplants as tooth substitutions may have unequivocal focal points as contrasted and osseointegrated gadgets, because of their potential for periodontal tissue recovery. It is recommended that restorative achievement requires the capacity of a high extent of the developed cells to coordinate into another PDL. Future clinical utilization of ligaplants may stay away from bone uniting with its cost, bother, and uneasiness for the patient. Such common combined and embedded mooring may likewise be viable with supplementary expansion and improvement of the jawbones lodging the teeth. This may allow for dental maturation and progress. It may help in during orthodontic treatment. Authoritatively, ligaplants have the facility to incite the procedure of the innovative bone creation, when put in terminuses related to enormous periodontal bone imperfections. Thus, stem cell therapies hold good promise for the future employing cryopreservation to make these prospects available to all age patients for future regenerative therapies. Tooth transplantation with a twofold PDL incitement is truly outstanding in instances of its PDL repair capacity or limit. This intentional injury set off a mending cycle inside the PDL, which incorporates cell multiplication and separation.
4. Material/substance used with specification Preparation of temperature-responsive culture dishes Steps under preparation: 1. 2-propanaol monomer and N-isopropylacylamide mix were poured onto polystyrene culture dishes. 2. The dishes were exposed completely to an Area Beam Electron Processing System (ABEP) using electron ray irradiation. 3. The temperature sensitive polymer-grafted that is poly Nisopropylacrylamide, dishes were dipped in water to eradicate ungrafted monomer. Then these were sterilized using ethylene oxide solution.
Cells and cell culture methodology The materials used for cells and culture were Himeso Mesenchymal stem cells expansion medium Steps Materials used in combinations 1 Dulbecco's Modified Eagle Media (DMEM) with low glucose 2 Antimycotic 1OOX solution (Thermofisher Scientific 3 Cat No-15240062 4 Cat No-11965-092 (Gibco, Invitrogen) 5 Fetal bovine serum (FBS) 6 Cat No -10270106(Gibco, Invitrogen, Antibiotic
5. Main Mechanism In the methodology, the first step was Isolation of Stem cells derived from PDL; 1) The teeth extracted were used to obtain PDL tissue. The tissue specimens are subjected to the followings in the cell culture laboratory
[100 U/ml penicillin + 100Og/ml streptomycin + 0.5% amphotericin B (Gibco, New York, USA) +10 ml culture medium (Dulbecco's modified Eagle's medium [DMEM/F 12]: Gibco, New York, USA; pH 7.2) + 10% heat-inactivated fetal bovine serum] 2) All tissue culture is exposed to laminar flow hood with the help of aseptic technique. The tissue specimens were exposed to a 10% povidone-iodine solution for 1 min to disinfect. 3) 1 mn size tissues were created and placed in the 15ml centrifuge containing collagenase typel (1mg/ml) and dispase (2mg/ml). Centrifuge tube incubated for 45 min to 1 h. The humidified atmosphere with (95% + 5%) air and Co2 is used to incubate the culture plate at 37C. 4) The next step was characterization of Stem cells derived from PDL. Isolated stem cells were grown on 96 well microtitre plate was with (95% + 5%) air and Co2 is used to incubate the culture plate at 37C overnight.
6. Proposed work layout Subsequent steps for Immunocytochemistry included and the same has been included in the file of drawing with subsequent samples of preparation. A. Keep block for first 5 minutes with PolyExcel H202 (Peroxide Block) B. CD-73, CD-90, CD105,CD-146 for 45 min at RT (Primary Antibody). C. For next 10 minute for incubation cover the tissue sections with the help of PolyExcel Target Binder (PolyExcel Target Binder) D. Again for next 10 minute at room temperature to incubate the tissue cover the tissue sections with the help of PolyExcel PolyHRP (PolyExcel PolyHRP:) E. Again for next 5 minute at room temperature to incubate the tissue cover the tissue sections with StunnDAB (PolyExcel StunnDAB). F. Hematoxylin is used to incubate the tissue for a time at room temperature. After the isolation, the cells were observed under phase-contrast microscopy using an inverted microscope.
7. Application • The proposed work delas with biomedical enginnering and medical science technology of tooth replacement. • Human tissues have been designed uniquely. In the current times, there has arisen another applicationand aggressive approach that joins information from material science with cell science and medication. • These are called realms of tissue engineering science areas. These techniques have utilized biodegradable polymers to make frameworks into the cells, embedded to create tissues within the sight of development factors. • Additionally, tooth replacement in animal model is another dimension of the proposed work.
8. Uniqueness • With effective minimally invasive procedures, the progenitor cells in periodontal tissue can be expanded in vitro, providing a singular reservoir of stem cells.
* They are a viable alternative to bone marrow aspiration, as procurement of PDLSCs from premolars is less invasive and morbid procedure with minimal complications. • The present study findings suggest that PDLSCs can be isolated and cultured from periodontal ligament cells of extracted adult premolars by using CD45, CD73, CD90, CD105 and CD146 markers.
Claims (7)
1. The progenitor cells in periodontal tissue can be expanded in vitro and offers a singular pool of stem cells.
2. They are a viable alternative to bone marrow aspiration, as procurement of PDLSCs from premolars is less invasive and morbid procedure with minimal complications.
3. It relieves from the medical issues such as bone defects, missing tooth and gingival recession.
4. It exactly mimics the process of natural tooth roots insertion in active alveolar methodology.
5. The present work findings suggest that PDLSCs can be isolated and cultured from periodontal ligament cells of extracted adult premolars by using CD45, CD73, CD90, CD105 and CD146 markers.
6. The current work elicits that CD 73, CD 90, CD 105, CD 146 is a reliable significant positive led marker for the identification of periodontal ligament stem cells.
7. CD 45 is a reliable negative marker for the identification of periodontal ligament stem cells.
Sample Images 21 May 2021 2021102741
Figure-4: Positive Staining for CD90 (20X Figure-1: Scraping of PDL cells from extracted magnification) teeth
Figure-2: Colonies of PDLSCs formed from Figure-5: Positive Staining for CD105 (20X isolated PDLSCs at 14 days magnification)
Figure-3: Positive Staining for CD73 (20X Figure-6: Positive Staining for CD146 (20X magnification) magnification)
Figure-10: Positive Staining for CD90 in pulpal 2021102741
Figure-7: Negative Staining for CD45 (20X MSCs (20X magnification) magnification)
Figure 8: Positive Staining for CD73 in pulpal Figure-11: Positive Staining for CD105 in pulp al MSCs (20X magnification) MSCs (20X magnification)
Figure-9: Positive Staining for CD105 in pulpal MSCs (20X magnification)
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| AU2021102741A AU2021102741A4 (en) | 2021-05-21 | 2021-05-21 | A novel methodology for the production and inoculation of periodontal ligament stem cells from periodontal ligament fibers on titanium implants in an animal model |
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|---|---|---|---|
| AU2021102741A AU2021102741A4 (en) | 2021-05-21 | 2021-05-21 | A novel methodology for the production and inoculation of periodontal ligament stem cells from periodontal ligament fibers on titanium implants in an animal model |
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| AU2021102741A4 true AU2021102741A4 (en) | 2022-03-24 |
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