Regeneration of periodontal ligament fibers around mini dental implants and their attachment to the bone in an animal model: A radiographic and histological study

Abstract:

Background:

Tissue-engineered periodontal ligament (PDL) around a dental implant by using PDL stem cells (PDLSCs) may be useful in periodontal regeneration and can reduce or eliminate certain shortcomings of dental implants.

Materials and Methods

PDLSCs were isolated from extracted human PDL cells and cultured in a bioreactor. They were identified using markers CD45, CD73, CD90, CD105, and CD146. After the formation of multiple cellular layers, they were then attached on titanium mini dental implants and placed in rabbit tibia. The rabbits were sacrificed after 9 months, and the implants were analyzed histologically and radiographically by Cone beam computed tomography (CBCT).

Results:

Isolated PDLSCs obtained from human premolars showed a colony-forming ability on the 7^th day and 14^th day. Immunocytochemistry revealed that cells had taken up the adequate positive stains for primary antibodies CD73, CD90, CD105, and CD146 and negative staining for CD45. The histological sections obtained from sacrificed rabbits, when viewed under the light microscope, clearly showed the presence of PDL around dental implants. CBCT examination showed that the implant was well within the bone and did not migrate. The site appeared to be normal without any lytic changes in the bone.

Conclusion:

It can safely be postulated from the present study that tissue engineering of PDL can be achieved around dental implants using PDLSCs. Important inter-tissue interactions like the formation of a functional PDL around the implantation site, and induction of bone formation in the vicinity of the implants may be possible. Future research in humans is required for further research.

INTRODUCTION

Though the field of regenerative dentistry has experienced myriad changes and enhancements, its operation in the field of implant dentistry is rather minimal. What this means is that presently the implant inserts are being put in with the point of achieving bony attachment. This happens without offering any chances to the recovery of periodontium around the alveolar bony areas.[1] The tissue of the human body has been designed uniquely. In the current times, there has arisen another aggressive approach that joins collective information of materials with cells and drugs. These ways have employed biodegradable polymers to make fabrics into the cells, bedded to generate tissues and cells with the help of development factors.[2-4]

Two primary kinds of undifferentiated stem cells exist, early-stage embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs are the consequence of the medication of an egg by a sperm. On about the 5^th day, this collection of cells turns into a blastocyst, an embryonic structure that’s comprised of 50150 cells.[5,6] Pluripotent stem cells (PSCs) are those early-stage undifferentiated cells that are gathered after the 5^th day from the inner cell mass of this blastocyst. These PSCs do separate into any of the three kinds of cells that determine the main three microscopic layers: ectoderm, endoderm, and mesoderm. These undifferentiated cells are now called multipotent stem cells (MSCs) and are conformed by day 14. One MSC can conceivably separate into any and every one of the 220 kinds of specific cells that make up the human body.[2,7-9] Still, early-stage undifferentiated PSCs have a high eventuality for causing cancers; making them less appropriate to use than the MSCs.[10-13]

The regenerative remedial aspects of periodontal infection have a significant application in clinical periodontics. Customary recovery treatments, like guided tissue regeneration, application of enamel matrix derivatives, and application of different development factors, can to some extent clinically recover destructed periodontal tissue.[9,14,15] Still, issues in their clinical operation exist. The treatment aspects change incredibly, contingent on the types and systems of the defects, or how much viably important periodontal ligament (PDL) is actually present.[16] The mainstay of periodontal recovery or regenerative treatments is to control inflammation and amplify ASCs to help recover and regenerate new periodontal tissues. The much decreased regenerative limit might be credited to inflammatory mediators delivered during the chronic patient inflammation in periodontal tissues.

Scientific research and examinations of the past demonstrate that PDL cells have the possibility to shape mineralized structures in vitro.[17] Examples illustrating these bone-related markers include bone sialoprotein and osteocalcin.[18] PDL cells also have the ability to communicate and mediate bone hormonal regulatory responses.[19] Therefore, with all aspects considered, PDL cells incorporate millions of cells which are in a wide range of phases of division which confer an applicable control of PDL cells, which is abecedarian for periodontal tissue engineering. As of late, it has been reckoned that periodontal tissues contain immature PDLSCs. These intriguing tissues have the fitness to frame tooth supporting-such as tissue in vivo. The application of PDL undifferentiated cells might have a fairly satisfying applicability in periodontal regenerative treatment.[20-22]

The PDLSCs which share numerous characteristics akin to the marrow substance of the bone are inferred as mesenchymal stem cells. Both cell types have the similar articulation of surface antigens (e.g., STRO-1, group of separation (CD 73, CD90, CD105, etc.,) and osteoblast-affiliated genes (Runx-2, antacid phosphatase, osteonectin, and so on) and can separate into osteoblasts, adipocytes, and chondrocytes in-vitro.[23,24] Still, PDL cells have a many unequivocal rudiments like tendon-related gene expression (scleraxis and tenomodulin) and also, can frame PDL-like tissues upon transplantation in-vivo.[25,26,27] Likewise, the evaluation of various bioscaffold accoutrements could help with feting reasonable transporters to help with initiating antecedent cells and better coordinate them into the general climate to further develop PDL foundational microorganism-interposed periodontal tissue recovery.[27]

Ligaplants are purely unique. The PDL, when it’s attached to the constructural frame of the surface implant biomaterial, it forms a new reality nominated as a Ligaplant.[2] Not veritably long agone, lost teeth were replaced without considering the PDL into account. The end-useable implants are made of an ideal biomaterial that directly gets embedded or osseointegrated into the jawbones.[3] Before these strategies, all neighborhood bone loss in the dental alveolus, and by and large all the decreased bone quality in similar regions were treated by grafting and bone reconstruction.[4] Besides that, confined bone misalignment around the PDL structure speaks of a considerable clinical challenge.[5] It’s particularly true in the cases of gingival insufficiency where, maybe because of altered tissue engineering, further careful interventions are warranted.[6] So, to conquer this issue, an embeded framework with PDL tissue-actuating parcels may be vitally important. Authoritatively, ligaplants have the installation to incite the procedure of innovative bone creation, when put in areas related to periodontal bone defects.[7-9] Thus, the current radiographic and histological study was undertaken to see the rejuvenescence of PDL fibers around mini dental implants and their attachment to the bone in an animal-based model.

MATERIALS AND METHODS

This study was conducted in two leading dental tutoring institutions of India. The endpoint aim of this animal experimental bid was to study histologically and radiographically the regeneration of PDL fibers around mini dental implants and their attachment to the bone in a rabbit model. The study followed the Animal Research: Reporting of In vivo Experiments Essential 10 guidelines and was approved by the institutional ethical committee. The objectives of this study were to radiographically evaluate the formation of bone and/or lamina dura around the implant surface and to histologically evaluate the implant-bone interface with the PDL cells in a rabbit model.

The inclusion criteria of periodontal samples and animals in the study were (i) extracted premolar teeth form youthful healthy grown-ups (removed from orthodontic purposes) (ii) healthy adult rabbits bred for the study purpose. Exclusion criteria were (i) individuals with any other medical conditions, (ii) rabbits who didn’t survive the study period. The study was done in two phases. Phase-1 initially involved the identification and culture of PDL stem cells (PDLSCs). This was followed by phase-2 which involved the placement of these PDLSCs along with the implant placement in the femur of rabbit. After 6 months, the rabbits were sacrificed and the effect of PDL conformation around the implant was studied.

Phase-1 of the study

Tooth transplantation with a twofold PDL incitement is truly outstanding in cases of its PDL forming capacity or limits. Fourteen days before transplantation, the donor tooth was extracted and instantly seeded or re-implanted in its own alveolus. This purposeful injury set off a mending cycle inside the PDL, which incorporates cell addition and separation. Following 14 days, the in-vivo cell culture achieves its peak of movement, and the tooth is transplanted with a significant number of cells with total mobility added to its root by new Sharpey’s fibers. We employed tissue engineering approaches to create a cell culture of a false root using this model in its natural and clinical settings.

A mixture of N-isopropylacylamide monomer and 2-propanol was applied to polystyrene culture dishes. In addition, the dishes were subjected to complete electron shaft irradiation utilizing an Area Beam Electron Processing System. The temperature-responsive polymer-grafted (poly Nisopropylacrylamide) dishes were castrated by ethylene oxide after being soaked in cold water to eliminate ungrafted monomer. 1 The accoutrements used for cells and culture were Himeso Mesenchymal stem cells expansion medium Dulbecco s Modified Eagle Media (DMEM) with low glucose-Cat No-11965-092 (Gibco, Invitrogen), Fetal bovine serum-Cat No-10270106 (Gibco, Invitrogen, Antibiotic– Antimycotic 100X result (Thermofisher Scientific)-Cat No-15240062.

In the methodology, the first step was the insulation of Stem cells deduced from PDL. Before retrieving the tissue sample, individuals were instructed to mouthwash using a solution of 10% povidone-iodine. The teeth extracted were used to obtain PDL tissue. To help cellular growth, the tissue samples were later transported to the cell culture laboratory in a 10 ml culture medium (Dulbecco’s modified Eagle’s medium (DMEM/F-12) Gibco, New York, USA; pH 7.2) supplemented with 10 heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.5 amphotericin B (Gibco, New York. Tissue culture was handled aseptically inside a laminar inflow hood. The tissue samples were then cleaned and disinfected in a 10% povidone-iodine solution for 1 min before being rinsed twice with phosphate-softened saline and finally with culture medium. The tissue samples were also cut into 1 mm pieces and deposited in a 15 ml centrifuge with collagenase type-1 (1 mg/ml) and dispase (2 mg/ml) and placed in the centrifuge tube for 45 min to 1 h. The culture plate was incubated at 37°C in the humidified environment of 95% air and 5% CO₂. An inverted microscope was used to view the culture plates on a regular basis.

The next step was to characterize the stem cells derived from PDL. Obtained stem cells were cultivated on a 96-well microtitre plate and incubated overnight at 37°C in a humidified environment of 95% air and 5% CO₂. Fixation was also performed for 10 min with 4% paraformaldehyde. The subsequent way for immunocytochemistry included:

1. Peroxide Block Block for 5 min with PolyExcel H₂O₂

2. Primary Antibody CD-73, CD-146 at normal temperature for 45 min

3. PolyExcel Target Binder Cover the tissue sections with PolyExcel Target Binder and Incubate for 10 min at RT

4. PolyExcel PolyHRP Cover the tissue sections with PolyExcel PolyHRP and incubate for 10 min at RT

5. PolyExcel StunnDAB Cover the tissue sections with StunnDAB working and incubate it for 5 min at room temperature

6. Hematoxylin cover the tissue sections with Hematoxylin and incubate for the applicable time at room temperature.

After the insulation, the cells were inspected under phase-discrepancy microscopy using a reversed microscope. Powers used were-

1. 10X power

2. 40X power.

Phase-2 of the study

After evidence cells were grown in Himeso Mesenchymal stem cells expansion medium in 25 cm² Tissue culture beaker for animal study. Ketamine 100 mg/kg body and xylazine 10 mg/kg were used to induce the anesthesia. An Oryctolagus cuniculus model was used for the study. The sample was of 4 rabbits-2 each of 2 different species. The age of the rabbits was 6 months. The ligaplants and control implants were placed in Rabbit-Tibial or femoral diaphyseal bone [Figure 1] Radiographic evaluations were done for over 9 months to check for PDL fibers attachment to the bone conformation of lamella dura around implants. Microscopic evaluation, after 9 months, of bone around the implant was done. The rabbit was appertained for Cone ray reckoned tomography (CBCT) imaging series for assessment of bone vacuity for implant placement in the tibia bone. CBCT was performed with Kavo 3D OP Pro with FOV (field of view) of 8 cm × 15 cm. Data was acquired as a volume accession and reconstructed in multiple aeroplanes. Evaluation of soft tissues is kindly limited by bone algorithm processing. Images handed were by a 3D volume rendered and reconstructed images and cross-sectional imaging series (1 mm slice interval, 1 mm consistency). Radiographic findings were observed covered in 8 cm × 15 cm FOV Tibia and fibula bone. After that, the rabbit was offered and the implant placed was recaptured surgically, and the instance was transferred for histopathology examination.

Figure 1.

Implant placed in rabbit bone

RESULTS

Twelve maxillary first premolar teeth were obtained from 6 patients who underwent orthodontic therapy at our institution, and were used in the study. Four patients were females and two were males. The patients were healthy young adolescents with ages ranging from 15 years to 21 years. None of the patients had any relevant medical history which affected the study. PDLSCs isolated from extracted human teeth were used in this study were used. The cells were isolated and cultured. The cells have a long and thin fibroblastic spindle shape in general. The unique clonogenic activity of undifferentiated stem cells is one of the primary indicator properties, which is also established as a sensitive indicator of these cells. Isolated PDLSCs have shown colony-forming potential in the current investigation. Colonies of PDLSCs were generated after 7 days of plating at a low density from isolated PDLSCs [Figure 2a]. The outcome was visible at 14 days [Figure 2b], along with an increment in colony volume. Immunocytochemistry has disclosed that the cells had taken up enough staining, indicating that they were overall positive. Under ×20 magnification, The cells with in tissue samples exhibited a positive staining for primary antibodies CD73, CD90, CD105, and CD146, as shown in Figure 3a-d. As demonstrated in Figure 3e, it stained negatively for CD4 5.

Figure 2.

Colonies of PDLSCs formed from isolated PDLSCs at; (a) 7 days and; (b) 14 days. PDLSCs: Periodontal ligament stem cells

Figure 3.

Positive Staining for; (a) CD73; (b) CD90; (c) CD105; (d) CD146 and negative staining for; (e) CD45 at ×20 magnification

On receiving the bone specimen with implant, the implant-bone tissue was fixed in neutral buffered formalin. After 48 h the tissue was immersed in decalcifying agent consisting of 5% formic acid and 5% of nitric acid. The sample was suspended in a beaker with the help of gauze piece in approximately 100 ml of the decalcifying agent. The precise timing of the initiation of decalcification was recorded. The temperature and ph of solutions were measured regularly. The final note of decalcification was estimated using chemical tests. On decalcification, the samples were sectioned and separated from the implant. The samples were subjected to manual processing in ascending grades of alcohol, acetone, and xylene using the standard protocol. The sample was embedded in paraffin wax in such a way that the tissue next to implant which was separated was on the cutting surface so that we could analyze the changes seen next to implant. The wax blocks prepared in this manner was sectioned to a thickness of 43μ using semiautomatic microtome. The sections were further stained by Ehrlich Hematoxylin and eosin stain and then viewed under light microscope by oral pathologists and assessed for the formation of PDL and cementum [Figures 4a-c, 5a and b]. PDLSCs were abundantly and positively seen in the histological sections.

Figure 4.

Photomicrograph of the decalcified section of bone shows; (a) a-Collagen fiber formation next to implant (PDL), b-Bone, (×10, H and E); (b) c-Bone formation, d-Bone formation, and; (c) e-collagen fibers (×40, H and E). PDL: Periodontal ligament

Figure 5.

Photomicrograph of the decalcified section of bone immediate attached to the implant shows; (a) f-collagen fibers resembling PDL (×10, H and E) and; (b) g-Bone formation (×40, H and E). PDL: Periodontal ligament

CBCT was done postoperatively at 9 months of the site of surgery. The scans obtained are shown in Figure 6a and b. The impant was noted to be well within the bone and did not migrate. The site appeared to be normal without any lytic changes in the bone. The existence of PDL surrounding titanium implants is indeed an eye-catching discovery. The finding of a PDL around titanium implants opens up new avenues in dental implantology. It now appears conceivable to accomplish dental implant anchoring with a normal periodontium, replicating the optimal role of human teeth. Moreover, these observations lay the groundwork for future research into the regeneration of all periodontal apparatus, including PDL, cementum, and bone.

Figure 6.

CBCT of implant in situ in the rabbit bone. CBCT: Cone beam computed tomography

DISCUSSION

Tissue engineering-related treatment ways have evolved as of late. PDLSCs, in this aspect, enjoy myriad benefits. They are clinically manageable, are easy to control, have a minimal complaint development, and have capacities to go through desirous change into final tissues and cells.[25] The periodontal tissues play a pivotal role in tissue development and alveolar bone development. Investigations undertaken in the current time have proposed that MSCs in periodontium can separate into cementum instituter cells, bone architecture cells, and certain particular cells creating fibroblasts.[8,26,27] PDLSCs are positioned in a particular region in periodontal tissue and they possess specific positive ascribes and characteristics of MSCs. In the current study, PDL test cells were retrieved by scratching the root’s face, a technique recently used by Navabazam et al., Park et al., and Silvério et al. to regenerate the PDL.[28-30]

In the current research, authors encountered an unconventional, yet a vital issue of microbial pollution or contamination of samples. Tissue samples can be polluted at any or every of the following three stages: (i) when tissue is removed from the patient, (ii) when it is transported to the laboratory where the disquisition is to be done, and (iii) when the media is set up and the tissues are processed. It was suggested in a work by Wanichpakorn and Kedjarune-Laggat that the tissue test characteristics may be related to and altered by microbial contamination.[31] In our review, we used 10% povidone-iodine to overcome this problem. The tissue was placed in the predetermined potency of povidone-iodine for approximately 2 min. To inhibit the development of gram-positive microorganisms, 100 units/mL of penicillin were added. Likewise, 100 g/L streptomycins were given to limit the development of Gram-negative microbes. Amphotericin B was used as a possible anti-fungal agent.

One specific cell’s capacity to multiply and shape an enormous colony is named as clonogenic development. This is, as expressed above, conceivably one of the main attributes of the undifferentiated stem cell. The quantitative strategy to examine this development is named as a Clonogenic assay. It is a well-known in vitro quantitative strategy, which has been employed in a myriad of investigations relating to a wide range of stem cells.[32] The present study has therefore demonstrated that the colony-forming capacity of the PDLSCs is a characteristic feature.

MSCs exhibit a variety of nonspecific markers, including CD73 (5′-ribonucleotide phosphohydrolase), CD90 (Thy-1; thymocyte separation antigen 1), CD105 (endoglin), and CD146 (MelCAM/MUC18). MSCs can be identified by various conventional labeling methodologies. CD73 is a very specific significant marker, which is used to portray the MSC crowd. CD73, also known as NTSE (ecto-5’- nucleotidase), is a glycophosphatidylinositol (GPI) protected emulsifier that creates extracellular adenosine. Another thing to keep in mind is that no consistent representation of CD73 articulation is noted. This nonuniform arrangement of CD73 in MSCs might be connected to their varied reformative and regenerative properties. Our findings evidently showed a high level of CD73 in PDL cells.

CD90 is a glycoprotein subclass that is furthermore known as Thy 1 since it is a thymocyte antigen. It’s a 2535 kDa roundly N-glycosylated GPI-secured covered cell face protein. Itis perhaps the most well-known basic microorganism marker. Our findings indicate the expression of CD90 in PDL cells. CD105 is an oxygen-deprivation protein that is abundant in angiogenic endothelial cells that plays a critical role in angiogenesis. Our findings demonstrate that the protestation of CD105 in PDL cells was all over the place.

PDLSCs with genuine CD146 show more significant colony-forming productivity as well as osteogenic outcomes than negative cells. CD146 is a transmembrane glycoprotein which is also known as S-Endo 1-related antigen. It is well recognized by two distinct names: Carcinoma cell bond flyspeck (MCAM) and cell face glycoprotein MUC18. In the current review, we had the option of restricting CD146 ve mortal PDLSCs, the level of which was similar to another research.[33,34] As a result, our review is unique, having several investigations on mortal PDLSCs. It is critical that CD146, together with STRO-1, are two major mainly recognized and effectively differentiated MSC designations.

Even though STRO-1 is a cellmarker found on all clonogenic stromal precursors, we did not employ it in the current review. Although STRO-1 is a well-known and well-documented marker of MSCs, there is some uncertainty over its use for immunohistochemistry.[35] To demonstrate this, Hung and associates discovered that ‘Size settled undifferentiated organisms,’ that seem to be a particular population of mortal bone gist-determined juvenile microorganisms, are consistently negative for STRO-1.[36] As a result, the use of STRO-1 as a counteracting drug as an MSC marker is marred by disagreements and necessitates an initial re-evaluation. This would be the primary cause we opted to exterminate STRO-1 from our disquisition. Another factor worth emphasizing is the negative take-up of CD45. CD45 is a marker for unoccupied hematopoietic cells. It is related to the sign transduction pathway in hematopoiesis. Our findings revealed the location of CD45 labels in PDL cells, which adapted to prior tests.

Our research shows that PDL-inferred dental progenitor cells (DPCs) might actually be employed to recover autologous PDL tissues on titanium implant inserts in the jaw. Refined PDL-determined DPCs showed high expansion rates, clonogenicity, and fat, bone, and neural cell separation. Gault et al. have expressed that the ligaplant structure imitates the normal addition of tooth established in the alveolar bone.[37] Tissue-designed PDL has been acquired beginning from a defined pad of PDL cells developed on porous hydroxyapatite (HAP)-covered Titanium pins. Ripamonti in 2007 has stated that tissues expresses another cementum-like layer, which is very ordinarily noted in recovered or regenerated PDL.[38] The typical arrangement of these cells and fibers are shaped transversely on the nonmineralized peri-implant space. This obviously proposes a recommending osteogenic capability of the newly formed PDL. Factual connections among implant and adjoining tooth roots, by which PDL cells could violate, were not observed at all.[39]

As of now, to rehabilitate lost teeth without the PDL, implant inserts of a nonreactive biomaterial are unswervingly embedded into jawbones. In this conduct, the bone defects in poor-quality bone require bony recreation or reconstruction before implant placement. State that such confined bone defects around the implant outfit connote a clinical reconstructive challenge.[40] A similar observation is also done by.[41] A further, regularly noticed issue is the gingival breakdown, combined with the capacity to sanitize and keep up with similar cells in vitro, which requires further careful surgical interventions.[42] An implant frame that would incorporate a PDL with tissue-initiating parcels may alleviate these issues significantly. Therefore, in our current research, we have depicted the technical turn of events and the clinical application of purported” ligaplants,” the blend of PDL cells with implant biomaterial. Ligaplants have the capacity of growth factors delivered in bone marrow stromal cells, they control abidance, expansion, separation, and capacity of immature PDLSCs and similar cells in various periodontal bone defects.

Jahangiri et al. conducted a review to speculate on the likelihood of PDL aging on an implant face by approaching a tooth-to-implant contact while using orthodontics.[39] In their review, the maxillary premolars of six beagle dogs were treated similarly to our study. Following 14 days of repairing, hydroxyapatite (HA) covered titanium implants, 5 mm long and 3.3 mm in distance across, were set in extraction sites. Control implants were utilized on one side of the dental arch. Orthodontic tooth advancement was started after implant integration to bring the important premolar roots into touch with the implant insert. This was accomplished in four and a half months, as evidenced by radiographs. Tooth-to-implant insertion contact was maintained for a month and a half, following which the teeth were orthodontically separated from bone-implant contact. The animals were relinquished after two substantial lengths of change. Faxitron radiographs were used to assess the tests before histology. Stevenel’s Blue and Van Gieson stains were used to build up histology experiments, which were then subjected to focused light microscopy. In four of the six animals where tooth-to-implant contact was obtained, the histologic analysis indicated trade and course of action of PDL-like design with cell cementum on the implant insert shells. Contact was observed in the zones coronal to the PDL-like tissue, a sure sign to perceive PDL-like tissue and connective tissue that may commence from the coronal portion of an embedded implant. They deduced that in their nonhuman experiment model, the proximity of tooth-to-implant touch resulted in partial PDL regeneration on a bioactive bone-implant interface in four out of six animals. This is veritably like the model employed in the current review. We have tracked down similar issues in our review.

Previous examinations have shown that the main cells living in the PDL are well equipped for forming new cementum by embedding collagen fibers into uncovered root surfaces. These studies focused on the application of PDL cells that had been cultured in vitro. The findings revealed that these cells had the ability to maintain their functionality and totality, which prompted a novel connection arrangement.

CONCLUSION

PDLSCs perhaps be easily identified from PDL cells of uprooted adult premolars using CD45, CD73, CD90, CD105, and CD146 labels, according to the findings of this study. It can also be safely supposed that from the present study tissue engineering of the PDL has been achieved as an evidence-of-conception. We’ve observed important inter-tissue relations like the conformation of a functional PDL around the implantation point, and induction of the conformation of bone in the vicinity of the implants. The clinical operation of tissue-engineering implants with PDL-like tissue is anticipated to present an indispensable remedial system in dental implantology allowing for a more natural and stable tooth-like dentition for a lifelong continuance.

Ethical approval and guidelines

Guidelines followed– Animal Research: Reporting of In vivo Experiments Essential 10 guidelines. Ethical Approval– Given by Institutional animal ethics committee (IAEC).

Financial support and sponsorship

A one-sentence summary describing the key finding(s) from the study.

PDLSCs can be isolated and cultured from PDL cells of extracted adult premolars by using CD45, CD73, CD90, CD105 and CD146 markers and important intertissue 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 can be seen in a rabbit model.

Conflicts of interest

There are no conflicts of interest.